Enolase 1 (eno1) compositions and uses thereof

ABSTRACT

The invention provides compositions comprising Eno1 for delivery to a muscle. Further, the invention provides a method for normalizing blood glucose in a subject with elevated blood glucose, comprising administering to the subject enolase 1 (Eno1), thereby normalizing blood glucose in the subject. The invention also provides methods of treating one or more conditions including impaired glucose tolerance, insulin resistance, pre-diabetes, and diabetes, especially type 2 diabetes in a subject, comprising administering to the subject enolase 1 (Eno1), thereby treating the condition in the subject. In certain methods of the invention, the Eno1 is delivered to muscle.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/926,913 filed on Jan. 13, 2014, U.S. Provisional PatentApplication No. 62/009,783 filed on Jun. 9, 2014, and U.S. ProvisionalPatent Application No. 62/100,881 filed on Jan. 7, 2015, the contents ofeach of which are incorporated herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 119992_(—)10604_Sequence_Listing. The size ofthe text file is 33 KB, and the text file was created on Jan. 13, 2015.

BACKGROUND

As the levels of blood glucose rise postprandially, insulin is secretedand stimulates cells of the peripheral tissues (skeletal muscles andfat) to actively take up glucose from the blood as a source of energy.Loss of glucose homeostasis as a result of dysregulated insulinsecretion or action typically results in metabolic disorders such asdiabetes, which may be co-triggered or further exacerbated by obesity.Because these conditions can reduce the quality of life or even befatal, strategies to restore adequate glucose clearance from thebloodstream are required.

Although diabetes may arise secondary to any condition that causesextensive damage to the pancreas (e.g., pancreatitis, tumors,administration of certain drugs such as corticosteroids or pentamidine,iron overload (i.e., hemochromatosis), acquired or geneticendocrinopathies, and surgical excision), the most common forms ofdiabetes typically arise from primary disorders of the insulin signalingsystem. There are two major types of diabetes, namely type 1 diabetes(also known as insulin dependent diabetes (IDDM)) and type 2 diabetes(also known as insulin independent or non-insulin dependent diabetes(NIDDM)), which share common long-term complications in spite of theirdifferent pathogenic mechanisms.

Type 1 diabetes, which accounts for approximately 10% of all cases ofprimary diabetes, is an organ-specific autoimmune disease characterizedby the extensive destruction of the insulin-producing beta cells of thepancreas. The consequent reduction in insulin production inevitablyleads to the deregulation of glucose metabolism. While theadministration of insulin provides significant benefits to patientssuffering from this condition, the short serum half-life of insulin is amajor impediment to the maintenance of normoglycemia. An alternativetreatment is islet transplantation, but this strategy has beenassociated with limited success.

Type 2 diabetes, which affects a larger proportion of the population, ischaracterized by a deregulation in the secretion of insulin and/or adecreased response of peripheral tissues to insulin, i.e., insulinresistance. While the pathogenesis of type 2 diabetes remains unclear,epidemiologic studies suggest that this form of diabetes results from acollection of multiple genetic defects or polymorphisms, eachcontributing its own predisposing risks and modified by environmentalfactors, including excess weight, diet, inactivity, drugs, and excessalcohol consumption. Although various therapeutic treatments areavailable for the management of type 2 diabetes, they are associatedwith various debilitating side effects. Accordingly, patients diagnosedwith or at risk of having type 2 diabetes are often advised to adopt ahealthier lifestyle, including loss of weight, change in diet, exercise,and moderate alcohol intake. Such lifestyle changes, however, are notsufficient to reverse the vascular and organ damages caused by diabetes.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compositions comprising enolase 1(Eno1) or a fragment thereof for delivery to a muscle. In one aspect,the invention provides method for normalizing blood glucose in a subjectwith elevated blood glucose, comprising administering to the subject acomposition comprising Eno1 or a fragment thereof, thereby normalizingblood glucose in the subject. In one aspect, the invention providesmethods of treating one or more conditions including impaired glucosetolerance, insulin resistance, pre-diabetes, and diabetes, especiallytype 2 diabetes, in a subject, comprising administering to the subject acomposition comprising Eno1 or a fragment thereof, thereby treating thecondition in the subject. In certain methods of the invention, the Eno1is delivered to muscle.

The invention provides pharmaceutical composition comprising Eno1 or afragment thereof for delivery to a muscle cell.

In certain embodiments, the Eno1 comprises an Eno1 polypeptide, or afragment thereof. In certain embodiments, the Eno1 comprises an Eno1nucleic acid, or a fragment thereof. In certain embodiments, the Eno1comprises human Eno1, e.g., a human Eno1 polypeptide or human Eno1nucleic acid, or fragment thereof.

In certain embodiments, the composition further comprises amicroparticle. In certain embodiments, the composition further comprisesa nanoparticle. In certain embodiments, the Eno1 or the fragment thereofis biologically active. In certain embodiments, the Eno1 or the fragmentthereof has at least 90% of the activity of a purified endogenous humanEno1 polypeptide.

In certain embodiments, the composition further comprises an in situforming composition. In certain embodiments, the composition furthercomprises a liposome. In certain embodiments, the composition comprisesa dendrimer. In certain embodiments, the composition further comprisesan expression vector, e.g., encoding the Eno1 or fragment thereof. Incertain embodiments, the expression vector comprises a viral vector.

In certain embodiments, the composition comprises a complex comprisingEno1 or a fragment thereof, e.g., an Eno1 polypeptide, e.g., a humanEno1 polypeptide, and a muscle targeting moiety. In certain embodiments,the muscle targeting moiety comprises a skeletal and/or smooth muscletargeting peptide). In certain embodiments, the MTP comprises an aminoacid sequence selected from the group consisting of: ASSLNIA; WDANGKT;GETRAPL; CGHHPVYAC; and HAIYPRH. In certain embodiments, the complexcomprises a linker, e.g., linking Eno1 and the SMTP. In certainembodiments, the linker is selected from the group consisting of acovalent linker, a non-covalent linkage, and a reversible linker. Incertain embodiments, the complex comprises a pharmaceutically acceptabledendrimer. In certain embodiments, the dendrimer is a PAMAM dendrimer.In certain embodiments, the dendrimer is a G5 dendrimer. In certainembodiments, the dendrimer is an uncharged dendrimer. In certainembodiments, the dendrimer is an acylated dendrimer. In certainembodiments, the dendrimer is a PEGylated dendrimer or an acetylateddendrimer. In certain embodiments, the complex comprises a liposome. Incertain embodiments, the complex comprises a microparticle or ananoparticle. In certain embodiments, the composition comprises an insitu forming composition.

In certain embodiments, the Eno1 is released from the complex upondelivery to a muscle cell.

In certain embodiments, the Eno1 or a fragment thereof and the targetingmoiety are present in the complex at a ratio of about 1:1 to about 1:30.

In certain embodiments, the composition is formulated for administrationby injection or infusion. In certain embodiments, the composition isformulated for oral administration. In certain embodiments, thecomposition is formulated for parenteral administration. In certainembodiments, the composition is formulated for intramuscularadministration, intravenous administration, or subcutaneousadministration.

The invention provides methods of decreasing blood glucose in a subjectwith elevated blood glucose, the method comprising administering to thesubject a pharmaceutical composition comprising of Eno1 or a fragmentthereof. In certain embodiments, the pharmaceutical compositioncomprises any of the pharmaceutical compositions provided herein.

The invention provides methods of increasing glucose tolerance in asubject with decreased glucose tolerance, the method comprisingadministering to the subject a pharmaceutical composition comprising ofEno1. In certain embodiments, the pharmaceutical composition comprisesany of the pharmaceutical compositions provided herein.

The invention provides methods of improving insulin response in asubject with decreased insulin sensitivity and/or insulin resistance,the method comprising administering to the subject a pharmaceuticalcomposition comprising of Eno1 or a fragment thereof. In certainembodiments, the pharmaceutical composition comprises any of thepharmaceutical compositions provided herein.

The invention provides methods of treating diabetes in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising of Eno1 or a fragment thereof. In certainembodiments, the diabetes is type 2 diabetes. In certain embodiments,the diabetes is pre-diabetes. In certain embodiments, the diabetes istype 1 diabetes. In certain embodiments, the diabetes is gestationaldiabetes. In certain embodiments, the pharmaceutical compositioncomprises any of the pharmaceutical compositions provided herein.

The invention provides methods of decreasing an HbA1c level in a subjectwith an elevated Hb1Ac level, the method comprising administering to thesubject a pharmaceutical composition comprising of Eno1 or a fragmentthereof. In certain embodiments, the pharmaceutical compositioncomprises any of the pharmaceutical compositions provided herein.

The invention provides methods of improving blood glucose level controlin a subject with abnormal blood glucose level control, the methodcomprising administering to the subject a pharmaceutical compositioncomprising of Eno1 or a fragment thereof. In certain embodiments, thepharmaceutical composition comprises any of the pharmaceuticalcompositions provided herein.

In certain embodiments, the Eno1 or a fragment thereof is administeredby injection or infusion. In certain embodiments, the Eno1 or a fragmentthereof is administered parenterally. In certain embodiments the Eno1 ora fragment thereof is administered orally. In certain embodiments, theEno1 or a fragment thereof is administered by a route selected from thegroup consisting of intramuscular, intravenous, and subcutaneous.

The invention provides methods for diagnosing an elevate blood glucoselevel in a subject, comprising: (a) detecting the level of Eno1 in abiological sample of the subject, and (b) comparing the level of Eno1 inthe biological sample with a predetermined threshold value, wherein thelevel Eno1 below the predetermined threshold value indicates thepresence of elevated blood glucose in the subject. In certainembodiments, the methods further comprise detecting the level of one ormore diagnostic indicators of elevated blood glucose. In certainembodiments, the one or more additional diagnostic indicators ofelevated blood glucose is selected from the group consisting of HbA1c,fasting blood glucose, fed blood glucose, and glucose tolerance. Incertain embodiments, the biological sample is blood or serum. In certainembodiments, the level of Eno1 is determined by immunoassay or ELISA. Incertain embodiments, step (a) comprises (i) contacting the biologicalsample with a reagent that selectively binds to the Eno1 to form abiomarker complex, and (ii) detecting the biomarker complex. In certainembodiments, the reagent is an anti-Eno1 antibody that selectively bindsto at least one epitope of Eno1.

In certain embodiments, step (a) comprises determining the amount ofEno1 mRNA in the biological sample. In certain embodiments, anamplification reaction is used for determining the amount of Eno1 mRNAin the biological sample. In certain embodiments, the amplificationreaction is (a) a polymerase chain reaction (PCR); (b) a nucleic acidsequence-based amplification assay (NASBA); (c) a transcription mediatedamplification (TMA); (d) a ligase chain reaction (LCR); or (e) a stranddisplacement amplification (SDA). In certain embodiments, ahybridization assay is used for determining the amount of Eno1 mRNA inthe biological sample. In certain embodiments, an oligonucleotide thatis complementary to a portion of a Eno1 mRNA is used in thehybridization assay to detect the Eno1 mRNA.

In certain embodiments of the invention, diagnosis of elevated bloodglucose is diagnostic of a disease or condition selected from the groupconsisting of type 2 diabetes, pre-diabetes, gestational diabetes, andtype 1 diabetes.

The invention provides method for diagnosing the presence of elevatedblood glucose in a subject, comprising:

(a) contacting a biological sample with a reagent that selectively bindsto Eno1;

(b) allowing a complex to form between the reagent and Eno1;

(c) detecting the level of the complex, and

(d) comparing the level of the complex with a predetermined thresholdvalue, wherein the level of the complex above the predeterminedthreshold value indicates the subject is suffering from elevated bloodglucose. In certain embodiments, the reagent is an anti-Eno1 antibody.In certain embodiments, the antibody comprises a detectable label. Incertain embodiments, the step of detecting the level of the complexfurther comprises contacting the complex with a detectable secondaryantibody and measuring the level of the secondary antibody. In certainembodiments, the methods further comprise detecting the level of one ormore additional indicators of elevated blood glucose. In certainembodiments, the one or more additional indicators of blood glucose isselected from the group consisting of HbA1c level, fasting glucoselevel, fed glucose level, and glucose tolerance. In certain embodiments,the biological sample is blood or serum.

In certain embodiments of the invention, the level of the complex isdetermined by immunoassay or ELISA. In certain embodiments, the elevatedblood glucose is indicative of pre-diabetes, type 2 diabetes, type 1diabetes, or gestational diabetes. In certain embodiments, the methodfurther comprises administering a therapeutic regimen where thediagnosis indicates the presence of elevated blood glucose in thesubject, wherein the therapeutic regimen is selected from the groupconsisting of drug therapy and behavioral therapy, or a combinationthereof. In certain embodiments, the drug therapy comprises treatmentwith an agent selected from the group consisting of (a) a meglitinide,(b) a sulfonylurea, (c) a dipeptidy peptidase-4 (DPP-4) inhibitor, (d) abiguanide, (e) a thiazolidinediones, (f) an alpha-glucosidase inhibitor,(g) an amylin mimetic; (h) an incretin mimetics; (i) an isulin; and (j)any combination thereof.

In certain embodiments, any of the preceding methods further compriseselecting a subject suspected of having or being at risk of havingelevated blood glucose.

In certain embodiments, any of the preceding methods further compriseobtaining a biological sample from a subject suspected of having orbeing at risk of having elevated blood glucose.

In certain embodiments, any of the preceding methods further comprisecomparing the level of the one or more elevated blood glucose relatedindicators in the biological sample with the level of the one or moreelevated blood glucose related indicators in a control sample selectedfrom the group consisting of: a sample obtained from the same subject atan earlier time point than the biological sample, a sample from asubject with normal blood glucose, a sample from a subject withprediabetes, a sample from a subject with type 2 diabetes, a sample froma subject with gestational diabetes, and a sample from a subject withtype 1 diabetes.

The invention provides methods for monitoring elevated blood glucose ina subject, the method comprising:

(1) determining a level of Eno1 in a first biological sample obtained ata first time from a subject having elevated blood glucose;

(2) determining a level of Eno1 in a second biological sample obtainedfrom the subject at a second time, wherein the second time is later thanthe first time; and

(3) comparing the level of Eno1 in the second sample with the level ofEno1 in the first sample, wherein a change in the level of Eno1 isindicative of a change in elevated blood glucose status in the subject.

In certain embodiments, the determining steps (1) and (2) furthercomprise determining the level of one or more additional indicators ofblood glucose is selected from the group consisting of HbA1c level,fasting glucose level, fed glucose level, and glucose tolerance.

In certain embodiments, the subject is treated with drugs for elevatedblood glucose prior to obtaining the second sample. In certainembodiments, a decreased level of Eno1 in the second biological sampleas compared to the first biological sample is indicative of elevation ofblood glucose in the subject. In certain embodiments, an increased orequivalent level of Eno1 in the second biological sample as compared tothe first biological sample is indicative of normalization of bloodglucose in the subject. In certain embodiments, the method furthercomprises selecting and/or administering a different treatment regimenfor the subject based on the blood glucose level in the subject. Incertain embodiments, the treatment regimen is selected from the groupconsisting of drug therapy and behavioral modification therapy. Incertain embodiments, the drug therapy comprises treatment with an agentselected from the group consisting of (a) a meglitinide, (b) asulfonylurea, (c) a dipeptidy peptidase-4 (DPP-4) inhibitor, (d) abiguanide, (e) a thiazolidinediones, (f) an alpha-glucosidase inhibitor,(g) an amylin mimetic; (h) an incretin mimetics; (i) an isulin; and (j)any combination thereof.

The invention provides methods of treating elevated blood glucose in asubject, comprising: (a) obtaining a biological sample from a subjectsuspected of having elevated blood glucose, (b) submitting thebiological sample to obtain diagnostic information as to the level ofEno1, (c) administering a therapeutically effective amount of ananti-diabetic therapy if the level of Eno1 is above a threshold level.

The invention provides methods of treating elevated blood glucose in asubject, comprising: (a) obtaining diagnostic information as to thelevel of Eno1 in a biological sample, and (b) administering atherapeutically effective amount of an anti-diabetic therapy if thelevel of Eno1 is above a threshold level.

The invention provides methods of treating elevated blood glucose in asubject, comprising:

(a) obtaining a biological sample from a subject suspected of havingelevated blood glucose for use in identifying diagnostic information asto the level of Eno1,

(b) measuring the level of Eno1 in the biological sample,

(c) recommending to a healthcare provider to administer a blood glucoselowering therapy if the level of Eno1 is below a threshold level.

In certain embodiments, the method further comprises obtainingdiagnostic information as to the level of one or more additionalindicators of elevated blood glucose.

In certain embodiments, the method further comprises measuring the levelof one or more additional indicators of elevated blood glucose.

In certain embodiments, the one or more additional indicators ofelevated blood glucose is selected from the group consisting of HbA1clevel, fasting glucose level, fed glucose level, and glucose tolerance.

In certain embodiments, step (c) further comprises administering atherapeutically effective amount of a glucose lowering therapy if thelevel of Eno1 is below and at least one of the additional indicator ofelevated blood glucose is detected. In certain embodiments, step (c)further comprises recommending to a healthcare provider to administer aglucose lowering therapy if the level of Eno1 is below a threshold leveland at least one of the additional indicator of elevated blood glucoseis present.

In certain embodiments, the biological sample is blood or serum. Incertain embodiments, the level of Eno1 is determined by immunoassay orELISA. In certain embodiments, the level of Eno1 is determined by (i)contacting the biological sample with a reagent that selectively bindsto the Eno1 to form a biomarker complex, and (ii) detecting thebiomarker complex. In certain embodiments, the reagent is an anti-Eno1antibody that selectively binds to at least one epitope of Eno1.

In certain embodiments, the level of Eno1 is determined by measuring theamount of Eno1 mRNA in the biological sample. In certain embodiments, anamplification reaction is used for measuring the amount of Eno1 mRNA inthe biological sample. In certain embodiments, the amplificationreaction is (a) a polymerase chain reaction (PCR); (b) a nucleic acidsequence-based amplification assay (NASBA); (c) a transcription mediatedamplification (TMA); (d) a ligase chain reaction (LCR); or (e) a stranddisplacement amplification (SDA). In certain embodiments, ahybridization assay is used for measuring the amount of Eno1 mRNA in thebiological sample. In certain embodiments, an oligonucleotide that iscomplementary to a portion of a Eno1 mRNA is used in the hybridizationassay to detect the Eno1 mRNA.

The invention provides kits for detecting Eno1 in a biological samplecomprising at least one reagent for measuring the level of Eno1 in thebiological sample, and a set of instructions for measuring the level ofEno1. In certain embodiments, the reagent is an anti-Eno1 antibody. Incertain embodiments, the kits further comprise a means to detect theanti-Eno1 antibody. In certain embodiments, the means to detect theanti-Eno1 antibody is a detectable secondary antibody. In certainembodiments, the reagent is an oligonucleotide that is complementary toa Eno1 mRNA. In certain embodiments, the instructions set forth animmunoassay or ELISA for detecting the Eno1 level in the biologicalsample. In certain embodiments, the instructions set forth anamplification reaction for assaying the level of Eno1 mRNA in thebiological sample. In certain embodiments, an amplification reaction isused for determining the amount of Eno1 mRNA in the biological sample.In certain embodiments, the amplification reaction is (a) a polymerasechain reaction (PCR); (b) a nucleic acid sequence-based amplificationassay (NASBA); (c) a transcription mediated amplification (TMA); (d) aligase chain reaction (LCR); or (e) a strand displacement amplification(SDA). In certain embodiments, the instructions set forth ahybridization assay for determining the amount of Eno1 mRNA in thebiological sample. In certain embodiments, the kit further comprises atleast one oligonucleotide that is complementary to a portion of a Eno1mRNA. In certain embodiments, the kit further comprises at least onereagent for measuring a level of HbA1c and/or blood glucose in thebiological sample. In certain embodiments, the kit further comprisesinstructions for measuring at least one level selected from the groupconsisting of HbA1c level, fed blood glucose level, fasting bloodglucose level, and glucose tolerance in the subject from which thebiological sample was obtained.

The invention provides panels of reagents for use in a method ofdetecting elevated blood glucose, the panel comprising detectionreagents for Eno1 and HbA1c.

The invention provides panels of reagents for use in a method oftreating elevated blood glucose, the panel comprising detection reagentsfor Eno1 and HbA1c.

The invention provides panels of reagents for use in a method ofmonitoring the treatment of elevated blood glucose, the panel comprisingdetection reagents for Eno1 and HbA1c.

The invention provides kits containing a the panel of reagents providedherein, and a set of instructions for obtaining diagnostic informationas to level of one or more indicators of elevated blood glucose.

The invention provides for the use of a panel of reagents comprising aplurality of detection reagents specific for detecting markers ofelevated blood glucose in a method for diagnosing and/or treatingelevated blood glucose, wherein at least one detection reagent of thepanel is specific for detecting Eno1, and wherein the remaining one ormore detection reagents are specific for detecting an indicator ofelevated blood glucose marker selected from the group consisting ofHbA1c and glucose.

In certain embodiments of the aforementioned methods, glucose flux in askeletal muscle cell of the subject is increased.

In another aspect, the invention provides a method of increasing glucoseflux in a subject, the method comprising administering to the subject apharmaceutical composition comprising Eno1 or a fragment thereof. Incertain embodiments, the pharmaceutical composition administered to thesubject is any of the aforementioned pharmaceutical compositions. Inanother aspect, the invention provides a method of increasing glycolyticactivity or capacity in a skeletal muscle cell of a subject, the methodcomprising administering to the subject a pharmaceutical compositioncomprising Eno1 or a fragment thereof. In certain embodiments, thepharmaceutical composition administered to the subject is any of theaforementioned pharmaceutical compositions.

In another aspect, the invention provides a method of increasingmitochondrial free fatty acid oxidation in a skeletal muscle cell of asubject, the method comprising administering to the subject apharmaceutical composition comprising Eno1 or a fragment thereof. Incertain embodiments, the pharmaceutical composition administered to thesubject is any of the aforementioned pharmaceutical compositions.

In certain embodiments, the subject has any one or more of elevatedblood glucose, decreased glucose tolerance, decreased insulinsensitivity and/or insulin resistance, diabetes, elevated Hb1Ac level,and abnormal blood glucose level control.

In certain embodiments of any of the aforementioned methods, the subjectis human.

In certain aspects the invention relates to a pharmaceutical compositioncomprising a therapeutically effective amount of Eno1 or a fragmentthereof. In certain embodiments, the pharmaceutical composition is fordelivery to a muscle cell. In certain embodiments of the composition,the Eno1 comprises an Eno1 polypeptide or a fragment thereof. In certainembodiments of the composition, the Eno1 comprises an Eno1 nucleic acidor a fragment thereof. In certain embodiments, the composition furthercomprises an expression vector encoding Eno1 or a fragment thereof. Incertain embodiments of the composition, the Eno1 or fragment thereof isbiologically active. In certain embodiments of the composition, the Eno1or fragment thereof has at least 90% of the activity of a purifiedendogenous human Eno1 polypeptide. In certain embodiments of thecomposition, the Eno1 is human Eno1. In certain embodiments, thecomposition further comprises a microparticle. In certain embodiments,the composition further comprises a nanoparticle. In certainembodiments, the composition further comprises an in situ formingcomposition. In certain embodiments, the composition further comprises aliposome. In certain embodiments, the composition further comprises amuscle targeting moiety. In certain embodiments, the muscle targetingmoiety is a skeletal muscle targeting moiety. In certain embodiments,the muscle targeting moiety and the Eno1 polypeptide are in a complex.

In certain embodiments of the compositions described herein, the Eno1 isreleased from the complex upon delivery to a muscle cell. In certainembodiments of the compositions described herein, the composition isformulated for parenteral administration. In certain embodiments, thecomposition is formulated for oral administration. In certainembodiments, the composition is formulated for intramuscularadministration, intravenous administration, or subcutaneousadministration.

In certain aspects the invention relates to a method of decreasing bloodglucose in a subject with elevated blood glucose, the method comprisingadministering to the subject a pharmaceutical composition comprisingEno1 or a fragment thereof, thereby decreasing blood glucose in thesubject. In certain embodiments of the aforementioned method, thepharmaceutical composition administered to the subject is any of thecompositions described above.

In certain aspects the invention relates to a method of increasingglucose tolerance in a subject with decreased glucose tolerance, themethod comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof, thereby increasingglucose tolerance in the subject. In certain embodiments of theaforementioned method, the pharmaceutical composition administered tothe subject is any of the compositions described above.

In certain aspects the invention relates to a method of improvinginsulin response in a subject with decreased insulin sensitivity and/orinsulin resistance, the method comprising administering to the subject apharmaceutical composition comprising Eno1 or a fragment thereof,thereby improving insulin response in the subject. In certainembodiments of the aforementioned method, the pharmaceutical compositionadministered to the subject is any of the compositions described above.

In certain aspects the invention relates to a method of treatingdiabetes in a subject, the method comprising administering to thesubject a pharmaceutical composition comprising Eno1 or a fragmentthereof, thereby treating diabetes in the subject. In certainembodiments, the diabetes is type 2 diabetes or type 1 diabetes. Incertain embodiments, the diabetes is pre-diabetes. In certainembodiments of the aforementioned method, the pharmaceutical compositionadministered to the subject is any of the compositions described above.

In certain aspects the invention relates to a method of decreasing anHbA1c level in a subject with an elevated Hb1Ac level, the methodcomprising administering to the subject a pharmaceutical compositioncomprising Eno1 or a fragment thereof, thereby decreasing the HbA1clevel in the subject. In certain embodiments of the aforementionedmethod, the pharmaceutical composition administered to the subject isany of the compositions described above.

In certain aspects the invention relates to a method of improving bloodglucose level control in a subject with abnormal blood glucose levelcontrol, the method comprising administering to the subject apharmaceutical composition comprising Eno1 or a fragment thereof,thereby improving blood glucose level control in the subject. In certainembodiments of the aforementioned method, the pharmaceutical compositionadministered to the subject is any of the compositions described above.

In certain embodiments of the aforementioned methods, the glucose fluxin a skeletal muscle cell of the subject is increased.

In certain aspects the invention relates to a method of increasingglucose flux in a subject, the method comprising administering to thesubject a pharmaceutical composition comprising Eno1 or a fragmentthereof, thereby increasing glucose flux in the subject. In certainembodiments of the aforementioned method, the pharmaceutical compositionadministered to the subject is any of the compositions described above.

In certain aspects the invention relates to a method of increasingglycolytic activity or capacity in a skeletal muscle cell of a subject,the method comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof, thereby increasingglycolytic activity or capacity in a skeletal muscle cell of thesubject. In certain embodiments of the aforementioned method, thepharmaceutical composition administered to the subject is any of thecompositions described above.

In certain aspects the invention relates to a method of increasingmitochondrial free fatty acid oxidation in a skeletal muscle cell of asubject, the method comprising administering to the subject apharmaceutical composition comprising Eno1 or a fragment thereof,thereby increasing mitochondrial free fatty acid oxidation in a skeletalmuscle cell of the subject. In certain embodiments of the aforementionedmethod, the pharmaceutical composition administered to the subject isany of the compositions described above.

In certain embodiments of the aforementioned methods, the Eno1 isadministered parenterally. In certain embodiments, the Eno1 isadministered orally. In certain embodiments, the Eno1 is administered bya route selected from the group consisting of intramuscular,intravenous, and subcutaneous. In certain embodiments, the subject hasany one or more of elevated blood glucose, decreased glucose tolerance,decreased insulin sensitivity and/or insulin resistance, diabetes,elevated Hb1Ac level, and abnormal blood glucose level control.

In certain embodiments of the aforementioned methods, the methodsfurther comprise selecting a subject having any one or more of elevatedblood glucose, decreased glucose tolerance, decreased insulinsensitivity and/or insulin resistance, diabetes, elevated Hb1Ac level,and abnormal blood glucose level control. In certain embodiments, thesubject is human.

In certain aspects the invention relates to a method for diagnosing anelevated blood glucose level in a subject, comprising: (a) detecting alevel of Eno1 in a biological sample from the subject, and (b) comparingthe level of Eno1 in the biological sample with a predeterminedthreshold value, wherein a level of Eno1 in the sample below thepredetermined threshold value indicates the presence of elevated bloodglucose in the subject. In certain embodiments, the method furthercomprises detecting the level of one or more diagnostic indicators ofelevated blood glucose. In certain embodiments, the one or moreadditional diagnostic indicators of elevated blood glucose is selectedfrom the group consisting of HbA1c, fasting blood glucose, fed bloodglucose, and glucose tolerance. In certain embodiments, the biologicalsample is blood or serum. In certain embodiments, the level of Eno1 isdetected by immunoassay or ELISA. In certain embodiments of theaforementioned method, step (a) comprises (i) contacting the biologicalsample with a reagent that selectively binds to the Eno1 to form abiomarker complex, and (ii) detecting the biomarker complex. In certainembodiments, the reagent is an anti-Eno1 antibody that selectively bindsto at least one epitope of Eno1. In certain embodiments, step (a)comprises detecting the amount of Eno1 mRNA in the biological sample. Incertain embodiments, an amplification reaction is used for detecting theamount of Eno1 mRNA in the biological sample. In certain embodiments,the amplification reaction is (a) a polymerase chain reaction (PCR); (b)a nucleic acid sequence-based amplification assay (NASBA); (c) atranscription mediated amplification (TMA); (d) a ligase chain reaction(LCR); or (e) a strand displacement amplification (SDA). In certainembodiments, a hybridization assay is used for detecting the amount ofEno1 mRNA in the biological sample. In certain embodiments, anoligonucleotide that is complementary to a portion of the Eno1 mRNA isused in the hybridization assay to detect the Eno1 mRNA.

In certain embodiments of the aforementioned method, the presence ofelevated blood glucose in the subject is diagnostic of a disease orcondition selected from the group consisting of type 2 diabetes,pre-diabetes, gestational diabetes, and type 1 diabetes.

In certain embodiments the aforementioned methods further compriseadministering a therapeutic regimen to the subject when the presence ofelevated blood glucose is determined, wherein the therapeutic regimen isselected from the group consisting of drug therapy and behavioraltherapy, or a combination thereof. In certain embodiments, the drugtherapy comprises treatment with an agent selected from the groupconsisting of (a) a meglitinide, (b) a sulfonylurea, (c) a dipeptidypeptidase-4 (DPP-4) inhibitor, (d) a biguanide, (e) athiazolidinediones, (f) an alpha-glucosidase inhibitor, (g) an amylinmimetic; (h) an incretin mimetics; (i) an insulin; and (j) anycombination thereof. In certain embodiments, the methods furthercomprise selecting a subject suspected of having or being at risk ofhaving elevated blood glucose. In certain embodiments the methodsfurther comprise obtaining a biological sample from a subject suspectedof having or being at risk of having elevated blood glucose.

In certain embodiments, the aforementioned methods further comprisecomparing the level of one or more elevated blood glucose relatedindicators in the biological sample with the level of the one or moreelevated blood glucose related indicators in a control sample selectedfrom the group consisting of: a sample obtained from the same subject atan earlier time point than the biological sample, a sample from asubject with normal blood glucose, a sample from a subject withprediabetes, a sample from a subject with type 2 diabetes, a sample froma subject with gestational diabetes, and a sample from a subject withtype 1 diabetes.

In certain aspects the invention relates to a method for monitoringelevated blood glucose in a subject, the method comprising: (1)determining a level of Eno1 in a first biological sample obtained at afirst time from a subject having elevated blood glucose; (2) determininga level of Eno1 in a second biological sample obtained from the subjectat a second time, wherein the second time is later than the first time;and (3) comparing the level of Eno1 in the second sample with the levelof Eno1 in the first sample, wherein a change in the level of Eno1 isindicative of a change in elevated blood glucose status in the subject.In certain embodiments, the determining steps (1) and (2) furthercomprise determining a level of one or more additional indicators ofblood glucose selected from the group consisting of HbA1c level, fastingglucose level, fed glucose level, and glucose tolerance. In certainembodiments, the subject is treated with drugs for elevated bloodglucose prior to obtaining the second sample. In certain embodiments, adecreased level of Eno1 in the second biological sample as compared tothe first biological sample is indicative of elevation of blood glucosein the subject. In certain embodiments, an increased or equivalent levelof Eno1 in the second biological sample as compared to the firstbiological sample is indicative of normalization of blood glucose in thesubject. In certain embodiments, the method further comprises selectingand/or administering a different treatment regimen for the subject basedon the blood glucose level in the subject. In certain embodiments, thetreatment regimen is selected from the group consisting of drug therapyand behavioral modification therapy. In certain embodiments, the drugtherapy comprises treatment with an agent selected from the groupconsisting of (a) a meglitinide, (b) a sulfonylurea, (c) a dipeptidypeptidase-4 (DPP-4) inhibitor, (d) a biguanide, (e) athiazolidinediones, (f) an alpha-glucosidase inhibitor, (g) an amylinmimetic; (h) an incretin mimetics; (i) an insulin; and (j) anycombination thereof.

In certain aspects the invention relates to a kit for detecting Eno1 ina biological sample comprising: (a) at least one reagent for measuringthe level of Eno1 in the biological sample; (b) a set of instructionsfor measuring the level of Eno1 in the biological sample; and (c) a setof instructions for determining the level of blood glucose in thebiological sample (e.g., based upon the level of Eno1). In certainembodiments the kit further comprises at least one reagent for measuringa level of HbA1c in the biological sample. In certain embodiments, thekit further comprises instructions for measuring at least one of HbA1clevel, fed blood glucose level, fasting blood glucose level, and glucosetolerance in the subject from which the biological sample was obtained.

In certain aspects the invention also relates to a panel of reagents foruse in a method of detecting elevated blood glucose, the panelcomprising detection reagents for Eno1 and HbA1c.

In certain aspects the invention also relates to a panel of reagents foruse in a method of treating elevated blood glucose, the panel comprisingdetection reagents for Eno1 and HbA1c.

In certain aspects the invention also relates to a panel of reagents foruse in a method of monitoring the treatment of elevated blood glucose,the panel comprising detection reagents for Eno1 and HbA1c.

In certain aspects the invention also relates to a kit comprising any ofthe panels of reagents described above, and a set of instructions forobtaining diagnostic information as to level of one or more indicatorsof elevated blood glucose.

In certain aspects the invention also relates to use of a panelcomprising a plurality of detection reagents specific for detectingmarkers of elevated blood glucose in a method for diagnosing and/ortreating elevated blood glucose, wherein at least one detection reagentof the panel is specific for detecting Eno1, and wherein the remainingone or more detection reagents are specific for detecting an indicatorof elevated blood glucose marker selected from the group consisting ofHbA1c and glucose.

Other embodiments are provided infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing glucose uptake in smooth muscle myoblaststreated with or without Eno1 and insulin. FIG. 1B shows glucose uptakein smooth muscle myoblasts treated with 0, 500, or 1000 ug/ml Eno1without insulin treatment.

FIGS. 2A and 2B show Eno1 protein levels in human skeletal musclemyotubes treated with 0, 500 or 1000 μg/ml Eno1. FIG. 2C shows Eno1activity in human skeletal muscle myotubes treated with 0, 500 or 1000μg/ml Eno1.

FIG. 3A shows Eno1 activity of native and heat inactivated Eno1. FIG. 3Bshows induction of glucose uptake by active and heat inactivated Eno1.

FIGS. 4A and 4B show (A) a time course and (B) the area under the curve(AUC) of glucose clearance in a glucose tolerance test in a mouse modelof diet induced obesity (DIO) after treatment with or without Eno1protein.

FIG. 5 shows Coomassie Staining of a polyacrylamide gel containingvarious concentrations of Eno1 analyzed by SDS-PAGE. L1: Precision PlusProtein Standard Dual Color, L2: Eno1 (10.0 μg), L3: Eno1 (1.0 μg), L4:Eno1 (0.1 μg).

FIG. 6 shows silver staining of a polyacrylamide gel containing variousconcentrations of Eno1 analyzed by SDS-PAGE. L1: Precision Plus ProteinStandard Dual Color, L2: Eno1 (10.0 μg), L3: Eno1 (1.0 μg), L4: Eno1(0.1 μg).

FIG. 7 shows Western Blot analysis of Eno1. L1: Precision Plus ProteinStandard Dual Color, L2: Eno1 (10.0 μg), L3: Eno1 (1.0 μg), L4: Eno1(0.1 μg).

FIG. 8 shows Zeta (ζ)-Potential measurement of Eno1/G5-dendrimer/SMTPcomplexes made with a 2:1 ratio of Eno1 to dendrimer SMTP.

FIG. 9 shows normalized activities of Eno1 alone (Enolase Alone) andEno1/G5-dendrimer/SMTP (Enolase/G5-SMC) solutions after storage atvarious temperatures.

FIGS. 10A and 10B are representative fluorescent images of the tissuedistribution in mice of (A) a fluorescently-labeled Eno1-G5-PAMAMdendrimer complex and (B) a fluorescently-labeled, muscle targetedEno-1-G5-PAMAM dendrimer complex.

FIG. 11 is a graph of the body weights of lean mice or DIO mice treatedwith one of vehicle, G5-PAMAM dendrimer, G5-PAMAM dendrimer+SMTP,G5-PAMAM dendrimer+Eno1, G5-PAMAM dendrimer+Eno1+SMTP.

FIG. 12 is a graph showing blood glucose levels in mice with dietinduced obesity after injection of saline or G5-PAMAMdendrimer+Eno1+SMTP (50 ug/kg) at 1, 4, and 24 hours after injection.

FIG. 13A shows the results of a glucose tolerance test in lean mice anda diet-induced obesity (DIO) mouse model of diabetes after 1 week oftreatment with G5-PAMAM dendrimer+SMTP (DIO NP+SMTP) or G5-PAMAMdendrimer+Eno1+SMTP (DIO Enolase-1+NP+SMTP). FIG. 13B shows the areaunder the curve (AUC) for each treatment group in FIG. 13A. FIGS. 13Cand 13D show (C) a time course and (D) the area under the curve (AUC) ofglucose clearance in a glucose tolerance test in lean mice or in DIOmice after two weeks treatment with one of vehicle, G5-PAMAM dendrimer(G5), G5-PAMAM dendrimer+SMTP, G5-PAMAM dendrimer+Eno1, G5-PAMAMdendrimer+Eno1+SMTP.

FIGS. 14A and 14B show (A) a time course and (B) the area under thecurve (AUC) of glucose clearance in a glucose tolerance test in leanmice or in DIO mice after four weeks treatment with one of vehicle,G5-PAMAM dendrimer, G5-PAMAM dendrimer+SMTP, G5-PAMAM dendrimer+Eno1,G5-PAMAM dendrimer+Eno1+SMTP.

FIG. 15 shows serum lactate levels in lean mice, diet induced obesity(DIO) mice, DIO mice treated with G5-dendrimer (DIO+NP), and DIO micetreated with Eno1/G5-dendrimer/SMTP complex (DIO+Eno-1+NP+SMTP) after 8weeks of treatment.

FIGS. 16A and 16B show (A) a time course and (B) the area under thecurve (AUC) of glucose clearance in an intraperitoneal glucose tolerancetest in db/db mice (BKS.Cg-m+/+Leprdb/J) after one week treatment withone of vehicle, G5-PAMAM dendrimer (G5)+SMTP, and G5-PAMAMdendrimer+Eno1+SMTP at 25 ug/kg or 50 ug/kg.

FIGS. 17A and 17B show a time course of glucose levels in db/db mice(BKS.Cg-m+/+Leprdb/J) after two weeks of treatment with one of vehicle,G5-PAMAM dendrimer (G5)+SMTP, and G5-PAMAM dendrimer+Eno1+SMTP at 25ug/kg or 50 ug/kg obtained in a time course initiated immediately afterinjection with the vehicle, G5-PAMAM dendrimer+SMTP, and G5-PAMAMdendrimer+Eno1+SMTP at the indicated doses. FIG. 17A shows the resultsfrom all four dosing regimens. FIG. 17B shows results from the G5-PAMAMdendrimer+SMTP (G5+SMTP) and G5-PAMAM dendrimer+Eno1+SMTP (Eno1+G5+SMTP)50 ug/kg to show the significant difference in glucose levels at the 30minute time point.

FIG. 18 shows the effect of once daily subcutaneous injection of 25μg/kg body weight or 50 μg/kg body weight of Eno1/G5-dendrimer/SMTPcomplex on fed blood glucose levels in a db/db diabetic mouse modelafter two weeks of treatment. Fed glucose was measured 24 hours afterEno1 injection without fasting. “NP” is the G5-dendrimer.

FIG. 19 shows the effect of twice daily (morning and evening)subcutaneous injection of 100 μg/kg body weight or 200 μg/kg body weightof Eno1/G5-dendrimer/SMTP complex on fed blood glucose levels in a db/dbdiabetic mouse model.

FIG. 20 shows creatine kinase and caspase 3 activity detected aftertreatment with G5-PAMAM dendrimer (G5), G5-PAMAM dendrimer+SMTP(G5-SMC), and acylated G5-PAMAM dendrimer+SMTP (G5-SMC-Ac).

FIG. 21 shows p-Akt protein levels in human skeletal muscle myotubeswith or without Eno1 and insulin treatment.

FIG. 22A shows Glut1, Glut4, HK2 and Myogenin mRNA levels in humanskeletal muscle myotubes with or without treatment with purified Eno1.FIG. 22B shows Glut1 protein levels in human skeletal muscle myotubeswith or without treatment with purified Eno1. Glut 1 protein levels arerelative units normalized by the ribosomal proteins median.

FIG. 23 shows glucose-6-phosphate (G6P) levels in glucose starved (toppanel) and glucose stimulated (bottom panel) human skeletal musclemyotubes with or without treatment with purified Eno1.

FIG. 24 shows phosphoenol pyruvate (PEP) levels in glucose starved (toppanel) and glucose stimulated (bottom panel) human skeletal musclemyotubes with or without treatment with purified Eno1.

FIG. 25 shows the oxygen consumption rate (OCR) in human skeletal musclemyotube (HSMM) cultures treated sequentially with palmitate, CCCP andetomoxir with or without treatment with purified Eno1.

FIG. 26 shows the extracellular acidification rate (ECAR) in humanskeletal muscle myotube (HSMM) cultures treated sequentially withglucose, oligomycin and 2-DG with or without treatment with purifiedEno1.

FIG. 27A shows mitochondrial content in human skeletal muscle myotubestreated with 500 μg/ml or 1000 μg/ml Eno1 relative to untreated controlhuman skeletal muscle myotubes. Mitochondrial content was determined byadding Mitotracker Green, a green fluorescent mitochondrial stain, tothe cells after 48 hours of Eno1 treatment.

FIG. 27B shows mitochondrial reactive oxygen species (Mito-ROS)production in human skeletal muscle myotubes treated with 500 ug/ml or1000 μg/ml Eno1 relative to untreated control human skeletal musclemyotubes (Eno 1 0 ug/ml). Mito-ROS was determined by treating cells withDihydrorhodamin 123, an uncharged and nonfluorescent reactive oxygenspecies (ROS) indicator that can passively diffuse across membraneswhere it is oxidized to cationic rhodamine 123 which localizes in themitochondria and exhibits green fluorescence. After dihydrorhodamin 123treatment, myotubes were trypsinized, washed, and subjected to flowcytometry to determine Mito-ROS levels.

FIG. 28A shows 5′ AMP activated protein kinase (AMPK) and phosphorylatedAMPK (pAMPK) levels in skeletal muscle myotubes treated with 0, 500, or1000 μg/ml Eno1. Lamin A/C was used as the loading control.

FIG. 28B shows the ratio of pAMPK (p-AMPK) to AMPK in basal and serumstarved skeletal muscle myotubes treated with 0, 500, or 1000 μg/mlEno1.

FIG. 29 shows a schematic of a working model describing the potentialrole of Nampt in the mode of action for Eno1.

FIG. 30 shows Nampt activity in human skeletal muscle myotubes treatedwith 500 ug/ml or 1000 μg/ml Eno1 in differentiation medium for 48 hoursafter 4 days of differentiation relative to untreated control humanskeletal muscle myotubes (Eno 1 0 ug/ml).

FIG. 31 shows 2-DG uptake in serum starved human skeletal musclemyotubes treated with recombinant extracellular Nampt (eNampt).

FIG. 32 shows glucose uptake in human skeletal muscle myotube culturestreated with 0, 500 or 1000 μg/ml Eno1 in differentiation medium for 48hours after 4 days of differentiation in the presence or absence of theNampt inhibitor FK866. FK866 was added 24 hours after initiation of Eno1treatment, and the myotubes were treated with FK866 for 24 hours. 2-DGuptake was measured after 3 hours serum starvation. Nampt inhibition byFK866 abolished Eno1 induced glucose uptake.

FIG. 33 shows a schematic of the glycolysis pathway.

FIGS. 34A and 34B show the (A) amino acid (SEQ ID NO: 2) and (B) nucleicacid coding sequence (SEQ ID NO: 1) of human Eno1, variant 1 (NCBIAccession No. NM_(—)001428.3).

FIGS. 35A and 35B show the (A) amino acid (SEQ ID NO: 4) and (B) nucleicacid coding sequence (SEQ ID NO: 3) of human Eno1, variant 2 (NCBIAccession No. NM_(—)001201483.1). The human Eno1, variant 2 protein isalso referred to as MBP-1.

FIG. 36A shows the nucleic acid sequence of ENO2 mRNA (SEQ ID NO: 5).FIG. 36B shows the amino acid sequence of Eno2 (SEQ ID NO: 6).

FIGS. 37A and 37B show the nucleic acid sequences of variant 1 (SEQ IDNO: 7) and variant 2 (SEQ ID NO: 8), respectively, of ENO3 mRNA. FIG.37C shows isoform 1 of the Eno3 protein (SEQ ID NO: 9), which is encodedby both variant 1 and variant 2. FIG. 37D shows the nucleic acidsequence of variant 3 of ENO3 mRNA (SEQ ID NO: 10). FIG. 37E shows theamino acid sequence of isoform 2 of Eno3 (SEQ ID NO: 11), which isencoded by variant 3. Variant 3 of the ENO3 mRNA differs in the 5′ UTRand lacks two exons in the 5′ coding region compared to variant 1.Isoform 2 of the Eno3 protein is shorter than isoform 1, but has thesame N- and C-termini.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

A discovery platform technology was used to delineate distinct molecularsignatures that drive the pathophysiology of diabetes. Eno1 wasidentified through this discovery platform technology as a critical nodethat is significantly modulated in human primary in vitro models ofdiabetes. Subsequent in vitro and in vivo studies discussed hereinconfirmed a role for Eno1 in insulin dependent and independent glucoseuptake, glucose tolerance, insulin sensitivity, and/or diabetes, e.g.,type 1 diabetes, type 2 diabetes, pre-diabetes, and gestationaldiabetes. More specifically, treatment of human myotubes with Eno1protein was demonstrated to increase both insulin independent anddependent glucose uptake in myotubes, indicating a role for Eno1 in thetreatment of both type 1 and type 2 diabetes and in glucose uptake inboth the presence and the absence of insulin and/or insulin response.Further, administration of Eno1 protein, either alone or in the contextof a skeletal muscle targeted dendrimer, improved glucose tolerance in adiet induced obesity model in mice, and similar results are expected ingenetic models of both type 1 and type 2 diabetes. These resultsdemonstrate that Eno1 is effective in normalizing glucose and insulinresponse, and thus indicate that Eno1 is useful in improving glucosetolerance and increasing insulin sensitivity/decreasing insulinresistance, thereby treating diabetes.

I. DEFINITIONS

Enolase 1, (alpha), also known as ENO1L, alpha-enolase, enolase-alpha,tau-crystallin, non-neural enolase (NNE), alpha enolase like 1,phosphopyruvate hydratase (PPH), plasminogen-binding protein, MYCpromoter-binding protein 1 (MPB1), and 2-phospho-D-glyceratehydro-lyase, is one of three enolase isoenzymes found in mammals.Protein and nucleic acid sequences of human Eno1 isoforms are providedherein in FIGS. 34 and 35. The instant application provides human aminoacid and nucleic acid sequences for the treatment of human disease.However, it is understood that the compositions and methods of theinvention can be readily adapted for treatment of non-human animals byselection of an Eno1 of the species to be treated. Amino acid andnucleic acid sequences of Eno1 for non-human species are known in theart and can be found, for example, at ncbi.nlm.nih.gov/genbank/. In someembodiments, the Eno1 used in the compositions and methods of theinvention is a mammalian Eno1. In a preferred embodiment, the Eno1 ishuman Eno1.

As used herein, “administration of Eno1” unless otherwise indicated isunderstood as administration of either Eno1 protein or a nucleic acidconstruct for expression of Eno1 protein. In certain embodiments theEno1 protein can include an Eno1 protein fragment or a nucleic acid forencoding an Eno1 protein fragment. In certain embodiments,administration of Eno1 is administration of Eno1 protein. In certainembodiments, administration of Eno1 is administration of Eno1polynucleotide. Protein and nucleic acid sequences of human Eno1 areprovided herein. In certain embodiments, administration of Eno1comprises administration of the first variant or the second variant ofhuman Eno1. In certain embodiments, administration of Eno1 comprisesadministration of the first variant and the second variant of humanEno1. In certain embodiments, administration of Eno1 comprisesadministration of the first variant of human Eno1. In certainembodiments, administration of Eno1 comprises administration of thesecond variant of human Eno1. In certain embodiments, administration ofEno1 comprises administration of only the first variant of human Eno1.In certain embodiments, administration of Eno1 comprises administrationof only the second variant of human Eno1.

As used herein, “biologically active” refers to an Eno1 molecule orfragment thereof that has at least one activity of an endogenous Eno1protein. For example, in some embodiments, the biologically active Eno1molecule or fragment thereof catalyzes the dehydration of2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP). In someembodiments, the biologically active Eno1 molecule or fragment thereofcatalyzes the hydration of PEP to PGA. In some embodiments, thebiologically active Eno1 molecule or fragment thereof increases glucoseuptake by a cell, for example a muscle cell, preferably a skeletalmuscle cell. In some embodiments, the biologically active Eno1 moleculeor fragment thereof reduces blood glucose levels, e.g. fed blood glucoselevels or blood glucose levels in a glucose tolerance test. In someembodiments, the biologically active Eno1 molecule or fragment thereofbinds to Nampt, for example, extracellular Nampt (eNampt).

As used herein, “administration to a muscle”, “delivery to a muscle”, or“delivery to a muscle cell” including a skeletal muscle cell, smoothmuscle cell, and the like are understood as a formulation, method, orcombination thereof to provide an effective dose of Eno1 to a musclee.g., a muscle cell, to provide a desired systemic effect, e.g.,normalization of blood glucose in a subject with abnormal blood glucose,e.g., by increasing glucose tolerance and/or insulin sensitivity, ortreating diabetes. In certain embodiments, the Eno1 is formulated foradministration directly to, and preferably retention in, muscle. Incertain embodiments, the formulation used for administration directly tothe muscle (i.e., intramuscular administration) preferably a sustainedrelease formulation of the Eno1 to permit a relatively low frequency ofadministration (e.g., once per week or less, every other week or less,once a month or less, once every other month or less, once every threemonths or less, once every four months or less, once every five monthsor less, once every six months or less). In certain embodiments, theEno1 is linked to a targeting moiety to increase delivery of the Eno1 tomuscle so that the Eno1 need not be delivered directly to muscle (e.g.,is delivered subcutaneously or intravenously). It is understood thatadministration to muscle does not require that the entire dose of Eno1be delivered to the muscle or into muscle cells. In certain embodiments,at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35% of the Eno1 is delivered to muscle, preferablyskeletal muscle and/or smooth muscle. In certain embodiments, the amountof non-intramuscularly administered muscle-targeted Eno1 delivered to amuscle cell is about 1.5 or more times greater, 2 or more times greater,3 or more times greater, 4 or more times greater, 5 or more timesgreater, or 6 or more times greater than the amount of non-targeted Eno1delivered to muscle. In certain embodiments, the Eno1 is delivered toskeletal muscle. In certain embodiments, the Eno1 is delivered to smoothmuscle. In certain embodiments, the Eno1 is delivered to skeletal muscleand smooth muscle. In certain embodiments, is delivered preferentiallyor in greater amount to skeletal muscle as compared to smooth muscle. Incertain embodiments, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% orgreater of the Eno1 delivered to muscle is delivered to skeletal muscle.In certain embodiments, the Eno1 is not delivered to smooth muscle.Assays to determine the relative targeting of a payload by a targetingmoiety are known in the art and provided, for example, in Samoylova etal., 1999, Muscle Nerve, 22:460-466, incorporated herein by reference.

As used herein, a “muscle targeting moiety” includes, at least, a muscletargeting peptide (MTP), for example a skeletal and/or smooth muscletargeting peptide (SMTP). In certain embodiments, the targeting moietyinclude ligands to bind integrins αvβ5 or αvβ3 integrins. In certainembodiments, the targeting moiety includes a CD-46 ligand. In certainembodiments, the targeting moiety includes an adenovirus peton proteinoptionally in combination with an adenovirus 35 fiber protein. Incertain embodiments, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35% of muscle-targeted Eno1 isdelivered to muscle, in some embodiments preferably skeletal and/orsmooth muscle, by a muscle-targeting moiety. In certain embodiments, theamount of non-intramuscularly administered muscle-targeted Eno1delivered to a muscle cell is about 1.5 or more times greater, 2 or moretimes greater, 3 or more times greater, 4 or more times greater, 5 ormore times greater, or 6 or more times greater than the amount ofnon-targeted Eno1 delivered to muscle.

As used herein, a “muscle targeting peptide” or “MTP” is understood as apeptide sequence that increases the delivery of its payload (e.g., Eno1)to a muscle cell, preferably a skeletal and/or smooth muscle cell. MTPsare known in the art and are provided, for example, in U.S. Pat. No.6,329,501; US Patent Publication No. 20110130346; and Samoylova et al.,1999, Muscle and Nerve 22: 460-466, each of which is incorporated hereinin its entirety. In certain embodiments the MTP is a skeletal muscletargeting peptide. A “skeletal muscle targeting peptide” is a peptidesequence that increases the delivery of its payload (e.g., Eno1) to askeletal muscle cell. In certain embodiments the MTP is a smooth muscletargeting peptide. A “smooth muscle targeting peptide” is a peptidesequence that increases the delivery of its payload (e.g., Eno1) to asmooth muscle cell. In certain embodiments the MTP increases thedelivery of its payload (e.g., Eno1) to a skeletal cell and to a smoothmuscle cell. In certain embodiments the MTP, e.g., skeletal muscletargeting peptide and/or smooth muscle targeting peptide, does notincrease the delivery of its payload to cardiac muscle cell. MTP, e.g.,skeletal muscle, targeting peptides include, but are not limited topeptides comprising the following sequences: ASSLNIA; WDANGKT; GETRAPL;CGHHPVYAC; and HAIYPRH. In a preferred embodiment, the MTP comprises theamino acid sequence ASSLNIA.

As used herein, “payload” is understood as a moiety for delivery to atarget cell by a targeting moiety. In certain embodiments, the payloadis a peptide, e.g., an Eno1 peptide. In certain embodiments, the payloadis a nucleic acid, e.g., a nucleic acid encoding an Eno1 peptide. Incertain embodiments, the payload further comprises additional components(e.g., dendrimers, liposomes, microparticles) or agents (e.g.,therapeutic agents) for delivery with the Eno1 payload to the targetcell.

As used herein, a “linker” is understood as a moiety that juxtaposes atargeting moiety and a payload in sufficiently close proximity such thatthe payload is delivered to the desired site by the targeting moiety. Incertain embodiments, the linker is a covalent linker, e.g., across-linking agent including a reversible cross-linking agent; apeptide bond, e.g., wherein the payload is a protein co-translated withthe targeting moiety. In certain embodiments, the linker is covalentlyjoined to one of the payload or the targeting moiety and non-covalentlylinked to the other. In certain embodiments, the linker comprises adendrimer. In certain embodiments, the dendrimer is covalently linked tothe targeting moiety and non-covalently linked to the payload, e.g.,Eno1. In certain embodiments, the linker is a liposome or amicroparticle, and the targeting moiety is exposed on the surface of theliposome and the payload, e.g., Eno1 is encapsulated in the liposome ormicroparticle. In certain embodiments, the linker and the Eno1 arepresent on the surface of the microparticle linker. In certainembodiments, the targeting moiety is present on the surface of a virusparticle and the payload comprises a nucleic acid encoding Eno1.

As used herein, “linked”, “operably linked”, “joined” and the like referto a juxtaposition wherein the components described are present in acomplex permitting them to function in their intended manner. Thecomponents can be linked covalently (e.g., peptide bond, disulfide bond,non-natural chemical linkage), through hydrogen bonding (e.g.,knob-into-holes pairing of proteins, see, e.g., U.S. Pat. No. 5,582,996;Watson-Crick nucleotide pairing), or ionic binding (e.g., chelator andmetal) either directly or through linkers (e.g., peptide sequences,typically short peptide sequences; nucleic acid sequences; or chemicallinkers, including the use of linkers for attachment to higher order orlarger structures including microparticles, beads, or dendrimers). Asused herein, components of a complex can be linked to each other bypackaging in and/or on a liposome and/or dendrimer wherein some of thecomponents of the complex can be attached covalently and somenon-covalently. Linkers can be used to provide separation between activemolecules so that the activity of the molecules is not substantiallyinhibited (less than 10%, less than 20%, less than 30%, less than 40%,less than 50%) by linking the first molecule to the second molecule.Linkers can be used, for example, in joining Eno1 to a targeting moiety.As used herein, molecules that are linked, but no covalently joined,have a binding affinity (Kd) of less than 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹², or any range bracketed by thosevalues, for each other under conditions in which the reagents of theinvention are used, i.e., typically physiological conditions.

In certain embodiments, the payload and the targeting moiety are presentin a complex at about a 1:1 molar ratio. In certain embodiments, thetargeting moiety is present in a complex with a molar excess of thepayload. In certain embodiments, the ratio of payload to targetingmoiety is about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1,about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, orabout 20:1.

A “dendrimer” is a polymeric molecule composed of multiple branchedmonomers that eminate radially from a central core. Due to the structureand synthetic methods used to generate dendrimers, the products fromdendrimer synthesis are theoretically monodisperse. When the core of adendrimer is removed, a number of identical fragments called dendronsremain with the number of dendrons dependent on the multiplicity of thecentral core. The number of branch points encountered upon movingoutward from the core to the periphery defines its generation, e.g.,G-1, G-2, G-3, etc., with dendrimers of higher generations being larger,more branched, and having more end groups than dendrimers of lowergenerations. As used herein, a dendrimer is preferably apharmaceutically acceptable dendrimer.

As used herein, a “subject with elevated blood glucose” or “increasedblood glucose” is understood as a subject who has elevated blood glucosefor a sufficient duration and frequency to be considered a pathologicalcondition, i.e., a subject that does not produce enough insulin or isnot sufficiently sensitive to insulin so that the glucose level of thesubject remains elevated for an extended period after eating a meal,e.g. for more than two hours after eating a meal and/or who has anelevated fasting blood glucose. In certain embodiments, a subject withelevated blood glucose is understood as a subject with one or both offasting blood glucose of at least 100 mg/dl and 2-hour plasma glucose ina 75-g oral glucose tolerance test of at least 140 mg/dl. In certainembodiments, a subject with elevated blood glucose is understood as asubject with one or more of fasting blood glucose of at least 126 mg/dl;a 2-hour plasma glucose in a 75-g oral glucose tolerance test of atleast 200 mg/dl; or a random plasma glucose of at least 200 mg/dl. Incertain embodiments, a subject with elevated blood glucose is understoodas a pregnant subject with one or more of fasting blood glucose of atleast 92 mg/dl; a 1-hour plasma glucose in a 75-g oral glucose tolerancetest of at least 180 mg/dl; and a 2-hour plasma glucose in a 75-g oralglucose tolerance test of at least 153 mg/dl. In certain embodiments asused herein, a subject with elevated blood glucose does not includesubjects with type 1 diabetes or pancreatic disease that results in anabsolute insulin deficiency. In certain embodiments as used herein, asubject with elevated blood glucose includes subjects with type 1diabetes or pancreatic disease that results in an absolute insulindeficiency.

As used herein, a “subject with elevated HbA1c” or a “subject withelevated A1c” is understood as a subject with an HbA1c level of at least5.7%. In certain embodiments, the subject has an HbA1c level of at least6.5%.

As used herein, “diabetes” is intended to refer to either type 1diabetes or type 2 diabetes, or both type 1 and type 2 diabetes,optionally in combination with gestational diabetes. In certainembodiments, diabetes includes type 2 diabetes. In certain embodiments,diabetes does not include type 1 diabetes. In certain embodiments,diabetes includes gestational diabetes. In certain embodiments, diabetesdoes not include gestational diabetes. In certain embodiments, diabetesincludes pre-diabetes. In certain embodiments, diabetes does not includepre-diabetes. In certain embodiments, diabetes includes pre-diabetes,type 1 diabetes, and type 2 diabetes. In certain embodiments, diabetesincludes pre-diabetes and type 2 diabetes.

As used herein, “insulin resistance” and “insulin insensitivity” can beused interchangeably and refers to conditions, especially pathologicalconditions, wherein the amount of insulin is less effective at loweringblood sugar than in a normal subject resulting in an increase in bloodsugar above the normal range that is not due to the absence of insulin.Without being bound by mechanism, the conditions are typicallyassociated with a decrease in signaling through the insulin receptor.Typically, insulin resistance in muscle and fat cells reduces glucoseuptake and storage as glycogen and triglycerides, respectively. Insulinresistance in liver cells results in reduced glycogen synthesis and afailure to suppress glucose production and release into the blood.

Insulin resistance is often present in the same subject together with“insulin insufficiency”, which also results in an increase in bloodsugar, especially a pathological increase in blood sugar, above thenormal range that is not due to the absence of insulin. Insulininsufficiency is a condition related to a lack of insulin action inwhich insulin is present and produced by the body. It is distinct fromtype 1 diabetes in which insulin is not produced due to the lack ofislet cells.

For the purposes of the methods of the instant invention, it is notnecessary to distinguish if a subject suffers from insulinresistance/insensitivity, insulin insufficiency, or both.

The term “impaired glucose tolerance” (IGT) or “pre-diabetes” is used todescribe a person who, when given a glucose tolerance test, has a bloodglucose level that falls between normal and hyperglycemic, i.e., hasabnormal glucose tolerance, e.g., pathologically abnormal glucosetolerance. Such a person is at a higher risk of developing diabetesalthough they are not clinically characterized as having diabetes. Forexample, impaired glucose tolerance refers to a condition in which apatient has a fasting blood glucose concentration or fasting serumglucose concentration greater than 110 mg/dl and less than 126 mg/dl(7.00 mmol/L), or a 2 hour postprandial blood glucose or serum glucoseconcentration greater than 140 mg/dl (7.78 mmol/L) and less than 200mg/dl (11.11 mmol/L). Prediabetes, also referred to as impaired glucosetolerance or impaired fasting glucose is a major risk factor for thedevelopment of type 2 diabetes mellitus, cardiovascular disease andmortality. Much focus has been given to developing therapeuticinterventions that prevent the development of type 2 diabetes byeffectively treating prediabetes (Pharmacotherapy, 24:362-71, 2004).

As used herein, a “pathological” condition reaches a clinicallyacceptable threshold of disease or condition. A pathological conditioncan result in significant adverse effects to the subject, particularlyin the long term, if the condition is not resolved, e.g., blood glucoseand/or HbA1c levels are not normalized. Pathological conditions can bereversed by therapeutic agents, surgery, and/or lifestyle changes. Apathological condition may or may not be chronic. A pathologicalcondition may or may not be reversible. A pathological condition may ormay not be terminal.

“Hyperinsulinemia” is defined as the condition in which a subject withinsulin resistance, with or without euglycemia, in which the fasting orpostprandial serum or plasma insulin concentration is elevated abovethat of normal, lean individuals without insulin resistance (i.e., >100mg/dl in a fasting plasma glucose test or >140 mg/dl in an oral glucosetolerance test).

The condition of “hyperglycemia” (high blood sugar) is a condition inwhich the blood glucose level is too high. Typically, hyperglycemiaoccurs when the blood glucose level rises above 180 mg/dl. Symptoms ofhyperglycemia include frequent urination, excessive thirst and, over alonger time span, weight loss.

The condition of “hypoglycemia” (low blood sugar) is a condition inwhich the blood glucose level is too low. Typically, hypoglycemia occurswhen the blood glucose level falls below 70 mg/dl. Symptoms ofhypoglycemia include moodiness, numbness of the extremities (especiallyin the hands and arms), confusion, shakiness or dizziness. Since thiscondition arises when there is an excess of insulin over the amount ofavailable glucose it is sometimes referred to as an insulin reaction.

As used herein, an “HbA1c level” or “A1c level” is understood as ahemoglobin A1c (HbA1c) level determined from an HbA1c test, whichassesses the average blood glucose levels during the previous two andthree months. A person without diabetes typically has an HbA1c valuethat ranges between 4% and 6%. Prediabetes is characterized by apathological HbA1c level of 5.7% to 6.5%, with an Hb1Ac level greaterthan 6.5% being indicative of diabetes. Every 1% increase in HbA1creflects a blood glucose levels increases by approximately 30 mg/dL andincreased risk of complications due to persistent elevated bloodglucose. Preferably, the HbA1c value of a patient being treatedaccording to the present invention is reduced to less than 9%, less than7%, less than 6%, and most preferably to around 5%. Thus, the excessHbA1c level of the patient being treated (i.e., the Hb1Ac level inexcess of 5.7%) is preferably lowered by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more relative to such levels prior totreatment (i.e., pre-treatment level−post-treatment level/pre-treatmentlevel).

As used herein, the term “subject” refers to human and non-humananimals, including veterinary subjects. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, mice, rabbits, sheep, dog, cat, horse, cow,chickens, amphibians, and reptiles. In a preferred embodiment, thesubject is a human and may be referred to as a patient.

As used herein, the terms “treat,” “treating” or “treatment” refer,preferably, to an action to obtain a beneficial or desired clinicalresult including, but not limited to, alleviation or amelioration of oneor more signs or symptoms of a disease or condition, diminishing theextent of disease, stability (i.e., not worsening) state of disease,amelioration or palliation of the disease state. As used herein,treatment can include one or more of reduction of insulin resistance,increasing insulin sensitivity, decreasing insulin deficiency, improvingor normalizing HbAc1 levels, improving or normalizing blood glucoselevels (e.g., fed blood glucose levels, fasting blood glucose levels,glucose tolerance), and ameliorating at least one sign or symptom ofdiabetes. Therapeutic goals in the treatment of diabetes, including type2 diabetes, include HbAc1 levels<6.5%; blood glucose 80-120 mg/dl beforemeals; and blood glucose<140 mg/dl 2 hours after meals. Therapeuticgoals in the treatment of pre-diabetes include reduction of HbA1c, bloodglucose levels, and glucose response to normal levels. Treatment doesnot need to be curative or reach the ideal therapeutic goals oftreatment. Treatment outcomes need not be determined quantitatively.However, in certain embodiments, treatment outcomes can be quantitatedby considering percent improvement towards a normal value at the end ofa range. For example, metabolic syndrome is characterized by an excessof some measures (e.g., blood glucose levels, HbA1c levels) and adeficiency in other measures (e.g., insulin response). A subject with afasting blood glucose level of 150 mg/dl would have excess fasting bloodglucose of 50 mg/dl (150 mg/dl-100 mg/dl, the maximum normal bloodglucose level). Reduction of excess blood glucose by 20% would be an 10mg/dl reduction in excess blood glucose. Similar calculations can bemade for other values.

As used herein, “reducing glucose levels” means reducing excess ofglucose by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, ormore to achieve a normalized glucose level, i.e., a glucose level nogreater than 150 mg/dl. Desirably, glucose levels prior to meals arereduced to normoglycemic levels, i.e., between 150 to 60 mg/dL, between140 to 70 mg/dL, between 130 to 70 mg/dL, between 125 to 80 mg/dL, andpreferably between 120 to 80 mg/dL. Such reduction in glucose levels maybe obtained by increasing any one of the biological activitiesassociated with the clearance of glucose from the blood. Accordingly, anagent having the ability to reduce glucose levels may increase insulinproduction, secretion, or action. Insulin action may be increased, forexample, by increasing glucose uptake by peripheral tissues and/or byreducing hepatic glucose production. Alternatively, the agent may reducethe absorption of carbohydrates from the intestines, alter glucosetransporter activity (e.g., by increasing GLUT4 expression, intrinsicactivity, or translocation), increase the amount of insulin-sensitivetissue (e.g., by increasing muscle cell or adipocyte celldifferentiation), or alter gene transcription in adipocytes or musclecells (e.g., altered secretion of factors from adipocytes expression ofmetabolic pathway genes). Desirably, the agent increases more than oneof the activities associated with the clearance of glucose.

By “alter insulin signaling pathway such that glucose levels arereduced” is meant to alter (by increasing or reducing) any one of theactivities involved in insulin signaling such that the overall result isan increase in the clearance of glucose from plasma and normalizes bloodglucose. For example, altering the insulin signaling pathway therebycausing an increase in insulin production, secretion, or action, anincreasing glucose uptake by peripheral tissues, a reducing hepaticglucose production, or a reducing the absorption of carbohydrates fromthe intestines.

A “therapeutically effective amount” is that amount sufficient to treata disease in a subject. A therapeutically effective amount can beadministered in one or more administrations.

A number of treatments for type 2 diabetes are known in the artincluding both drug and behavioral interventions. Drugs for treatment oftype 2 diabetes include, but are not limited to meglitinides(repaglinide (Prandin) and nateglinide (Starlix); sulfonylureas(glipizide (Glucotrol), glimepiride (Amaryl), and glyburide (DiaBeta,Glynase)); Dipeptidy peptidase-4 (DPP-4) inhibitors (saxagliptin(Onglyza), sitagliptin (Januvia), and linagliptin (Tradjenta));biguanides (metformin (Fortamet, Glucophage)); thiazolidinediones(rosiglitazone (Avandia) and pioglitazone (Actos)); andalpha-glucosidase inhibitors (acarbose (Precose) and miglitol (Glyset)).Insulins are typically used only in treatment of later stage type 2diabetes and include rapid-acting insulin (insulin aspart (NovoLog),insulin glulisine (Apidra), and insulin lispro (Humalog)); short-actinginsulin (insulin regular (Humulin R, Novolin R)); intermediate-actinginsulin (insulin NPH human (Humulin N, Novolin N)), and long-actinginsulin (insulin glargine (Lantus) and insulin detemir (Levemir)).Treatments for diabetes can also include behavior modification includingexercise and weight loss which can be facilitated by the use of drugs orsurgery. Treatments for elevated blood glucose and diabetes can becombined. For example, drug therapy can be combined with behaviormodification therapy.

By “diagnosing” and the like, as used herein, refers to a clinical orother assessment of the condition of a subject based on observation,testing, or circumstances for identifying a subject having a disease,disorder, or condition based on the presence of at least one indicator,such as a sign or symptom of the disease, disorder, or condition.Typically, diagnosing using the method of the invention includes theobservation of the subject for multiple indicators of the disease,disorder, or condition in conjunction with the methods provided herein.Diagnostic methods provide an indicator that a disease is or is notpresent. A single diagnostic test typically does not provide adefinitive conclusion regarding the disease state of the subject beingtested.

As used herein, “monitoring” is understood as assessing at least onesign or symptom of a disease in a subject at a first time point and at alater second time point, comparing the severity of the sign(s) orsymptom(s) of the condition, and determining of the condition becamemore or less severe over time.

The terms “administer”, “administering” or “administration” include anymethod of delivery of a pharmaceutical composition or agent into asubject's system or to a particular region in or on a subject. Incertain embodiments, the agent is administered enterally orparenterally. In certain embodiments of the invention, an agent isadministered intravenously, intramuscularly, subcutaneously,intradermally, intranasally, orally, transcutaneously, or mucosally. Incertain preferred embodiments, an agent is administered by injection orinfusion, e.g., intravenously, intramuscularly, subcutaneously. Incertain embodiments, administration includes the use of a pump. Incertain embodiments, the agent is administered locally or systemically.Administering an agent can be performed by a number of people working inconcert. Administering an agent includes, for example, prescribing anagent to be administered to a subject and/or providing instructions,directly or through another, to take a specific agent, either byself-delivery, e.g., as by oral delivery, subcutaneous delivery,intravenous delivery through a central line, etc.; or for delivery by atrained professional, e.g., intravenous delivery, intramusculardelivery, etc.

As used herein, the term “co-administering” refers to administration ofEno1 prior to, concurrently or substantially concurrently with,subsequently to, or intermittently with the administration of an agentfor the treatment of diabetes, pre-diabetes, glucose intolerance, orinsulin resistance. The Eno1 formulations provided herein, can be usedin combination therapy with at least one other therapeutic agent for thetreatment of diabetes, pre-diabetes, glucose intolerance, or insulinresistance. Eno1 and/or pharmaceutical formulations thereof and theother therapeutic agent can act additively or, more preferably,synergistically. In one embodiment, Eno1 and/or a formulation thereof isadministered concurrently with the administration of another therapeuticagent for the treatment of diabetes, pre-diabetes, glucose intolerance,or insulin resistance. In another embodiment, Eno1 and/or apharmaceutical formulation thereof is administered prior or subsequentto administration of another therapeutic agent for the treatment ofdiabetes, pre-diabetes, glucose intolerance, or insulin resistance.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject. The term “sample”includes any body fluid (e.g., urine, serum, blood fluids, lymph,gynecological fluids, cystic fluid, ascetic fluid, ocular fluids, andfluids collected by bronchial lavage and/or peritoneal rinsing),ascites, tissue samples or a cell from a subject. Other subject samplesinclude tear drops, serum, cerebrospinal fluid, feces, sputum, and cellextracts. In a particular embodiment, the sample is urine or serum. Incertain embodiments, the sample comprises cells. In other embodiments,the sample does not comprise cells.

The term “control sample,” as used herein, refers to any clinicallyrelevant comparative sample, including, for example, a sample from ahealthy subject not afflicted with any of impaired glucose tolerance,increased blood glucose, insulin resistance, diabetes, or prediabetes;or a sample from a subject from an earlier time point in the subject,e.g., prior to treatment, at an earlier stage of treatment. A controlsample can be a purified sample, protein, and/or nucleic acid providedwith a kit. Such control samples can be diluted, for example, in adilution series to allow for quantitative measurement of analytes intest samples. A control sample may include a sample derived from one ormore subjects. A control sample may also be a sample made at an earliertime point from the subject to be assessed. For example, the controlsample can be a sample taken from the subject to be assessed before theonset abnormal blood glucose levels or A1c levels, at an earlier stageof disease, or before the administration of treatment or of a portion oftreatment. The control sample may also be a sample from an animal model,or from a tissue or cell lines derived from the animal model of impairedglucose tolerance, increased blood glucose, insulin resistance,diabetes, or prediabetes. The level of Eno1 activity or expression in acontrol sample that consists of a group of measurements may bedetermined, e.g., based on any appropriate statistical measure, such as,for example, measures of central tendency including average, median, ormodal values.

The term “control level” refers to an accepted or pre-determined levelof a sign of a impaired glucose tolerance, increased blood glucose,insulin resistance, diabetes, or pre-diabetes in a subject or a subjectsample. The following levels are considered to be normal levels:

-   -   Fasting blood glucose less than or equal to 100 mg/dl.    -   HbA1c less than or equal to 5.7%.    -   Oral glucose tolerance test less than or equal to 140 mg/dl.

Levels above these levels are understood to be pathological levels.

As used herein, a “predetermined threshold value” of a biomarker refersto the level of the biomarker (e.g., the expression level or quantity(e.g., ng/ml) in a biological sample) or other indicator of elevatedblood glucose in a corresponding control/normal sample or group ofcontrol/normal samples obtained from normal or healthy subjects, e.g.,subjects that do not have abnormal blood glucose. The predeterminedthreshold value may be determined prior to or concurrently withmeasurement of marker levels in a biological sample. The control samplemay be from the same subject at a previous time or from differentsubjects.

As used herein, a sample obtained at an “earlier time point” is a samplethat was obtained at a sufficient time in the past such that clinicallyrelevant information could be obtained in the sample from the earliertime point as compared to the later time point. In certain embodiments,an earlier time point is at least four weeks earlier. In certainembodiments, an earlier time point is at least six weeks earlier. Incertain embodiments, an earlier time point is at least two monthsearlier. In certain embodiments, an earlier time point is at least threemonths earlier. In certain embodiments, an earlier time point is atleast six months earlier. In certain embodiments, an earlier time pointis at least nine months earlier. In certain embodiments, an earlier timepoint is at least one year earlier. Multiple subject samples (e.g., 3,4, 5, 6, 7, or more) can be obtained at regular or irregular intervalsover time and analyzed for trends in changes in marker levels.Appropriate intervals for testing for a particular subject can bedetermined by one of skill in the art based on ordinary considerations.

As used herein, the term “obtaining” is understood to refer tomanufacturing, purchasing, or otherwise coming into possession of.

As used herein, “detecting”, “detection” and the like are understood torefer to an assay performed for identification of a specific analyte ina sample, e.g., Eno1 expression or activity level in a sample. Theamount of analyte or activity detected in the sample can be none orbelow the level of detection of the assay or method. Detecting ordetection can also include measuring of glucose and/of HbAc1 levels.

The terms “modulate” or “modulation” refer to upregulation (i.e.,activation or stimulation), downregulation (i.e., inhibition orsuppression) of a level, or the two in combination or apart. A“modulator” is a compound or molecule that modulates, and may be, e.g.,an agonist, antagonist, activator, stimulator, suppressor, or inhibitor.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, or protein, or both.

The terms “level of expression of a gene” or “gene expression level”refer to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, or the level of protein, encoded by the gene in the cell.

As used herein, the term “amplification” refers to any known in vitroprocedure for obtaining multiple copies (“amplicons”) of a targetnucleic acid sequence or its complement or fragments thereof. In vitroamplification refers to production of an amplified nucleic acid that maycontain less than the complete target region sequence or its complement.Known in vitro amplification methods include, e.g.,transcription-mediated amplification, replicase-mediated amplification,polymerase chain reaction (PCR) amplification, ligase chain reaction(LCR) amplification and strand-displacement amplification (SDA includingmultiple strand-displacement amplification method (MSDA)).Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as Q-β-replicase (e.g., Kramer et al., U.S. Pat.No. 4,786,600). PCR amplification is well known and uses DNA polymerase,primers and thermal cycling to synthesize multiple copies of the twocomplementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses atleast four separate oligonucleotides to amplify a target and itscomplementary strand by using multiple cycles of hybridization,ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308). SDAis a method in which a primer contains a recognition site for arestriction endonuclease that permits the endonuclease to nick onestrand of a hemimodified DNA duplex that includes the target sequence,followed by amplification in a series of primer extension and stranddisplacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Twoother known strand-displacement amplification methods do not requireendonuclease nicking (Dattagupta et al., U.S. Pat. No. 6,087,133 andU.S. Pat. No. 6,124,120 (MSDA)). Those skilled in the art willunderstand that the oligonucleotide primer sequences of the presentinvention may be readily used in any in vitro amplification method basedon primer extension by a polymerase. (see generally Kwoh et al., 1990,Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad.Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202;Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al.,2000, Molecular Cloning—A Laboratory Manual, Third Edition, CSHLaboratories). As commonly known in the art, the oligos are designed tobind to a complementary sequence under selected conditions.

As used herein, the term “antigen” refers to a molecule, e.g., apeptide, polypeptide, protein, fragment, or other biological moiety,which elicits an antibody response in a subject, or is recognized andbound by an antibody.

As used herein, the term “complementary” refers to the broad concept ofsequence complementarity between regions of two nucleic acid strands orbetween two regions of the same nucleic acid strand. It is known that anadenine residue of a first nucleic acid region is capable of formingspecific hydrogen bonds (“base pairing”) with a residue of a secondnucleic acid region which is antiparallel to the first region if theresidue is thymine or uracil. Similarly, it is known that a cytosineresidue of a first nucleic acid strand is capable of base pairing with aresidue of a second nucleic acid strand which is antiparallel to thefirst strand if the residue is guanine. A first region of a nucleic acidis complementary to a second region of the same or a different nucleicacid if, when the two regions are arranged in an antiparallel fashion,at least one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

As use herein, the phrase “specific binding” or “specifically binding”when used in reference to the interaction of an antibody and a proteinor peptide means that the interaction is dependent upon the presence ofa particular structure (i.e., the antigenic determinant or epitope) onthe protein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

The phrase “specific identification” is understood as detection of amarker of interest with sufficiently low background of the assay andcross-reactivity of the reagents used such that the detection method isdiagnostically useful. In certain embodiments, reagents for specificidentification of a marker bind to only one isoform of the marker. Incertain embodiments, reagents for specific identification of a markerbind to more than one isoform of the marker. In certain embodiments,reagents for specific identification of a marker bind to all knownisoforms of the marker.

As used herein, the phrase “subject suspected of having elevated bloodglucose” refers to a subject that presents one or more signs or symptomsindicative of or correlated with elevated blood glucose or is beingscreened for a elevated blood glucose (e.g., during a routine physical).A subject suspected of having elevated blood glucose may also have oneor more risk factors. A subject suspected of having elevated bloodglucose has generally not been tested for abnormal glucose levels,metabolism, or response. However, a “subject suspected of havingelevated blood glucose” encompasses an individual who has received aninitial diagnosis (e.g., a single incidence of elevated, but notconfirmed, blood glucose) but for whom the degree of elevated glucose isnot known. The term further includes people who once had elevated bloodglucose (e.g., an individual treated for elevated blood glucose whomaintained a normal blood glucose and/or HbA1c levels for an extendedperiod, e.g., at least 3 months, at least 6 months, etc.).

The articles “a”, “an” and “the” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle unless otherwise clearly indicated by contrast. By way ofexample, “an element” means one element or more than one element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to”.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical group(s) in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that it is not intended tolimit the invention to those preferred embodiments. To the contrary, itis intended to cover alternatives, modifications, and equivalents as maybe included within the spirit and scope of the invention as defined bythe appended claims.

IIA. Enolase 1

Enolase 1, (alpha), also known as ENO1L, alpha-enolase, enolase-alpha,tau-crystallin, non-neural enolase (NNE), alpha enolase like 1,phosphopyruvate hydratase (PPH), plasminogen-binding protein, MYCpromoter-binding protein 1 (MPB1), and 2-phospho-D-glyceratehydro-lyase, is one of three enolase isoenzymes found in mammals. Eachisoenzyme is a homodimer composed of 2 alpha, 2 gamma, or 2 betasubunits, and functions as a glycolytic enzyme. Alpha-enolase inaddition, functions as a structural lens protein (tau-crystallin) in themonomeric form. Alternative splicing of this gene results in a shorterisoform that has been shown to bind to the c-myc promoter and functionas a tumor suppressor. Several pseudogenes have been identified,including one on the long arm of chromosome 1. Alpha-enolase has alsobeen identified as an autoantigen in Hashimoto encephalopathy. Furtherinformation regarding human Eno1 can be found, for example, in the NCBIgene database under Gene ID No. 2023 (see,www.ncbi.nlm.nih.gov/gene/2023, incorporated herein by reference in theversion available on the date of filing this application).

Eno1 Variants

Two isoforms of human Eno1 are known. Protein and mRNA sequences of Homosapiens enolase 1, (alpha) (ENO1), transcript variant 1, mRNA can befound at GenBank Accession No. NM_(—)001428 (seewww.ncbi.nlm.nih.gov/nuccore/NM_(—)001428.3, which is incorporated byreference in the version available on the date of filing the instantapplication). This variant encodes the longer isoform, which islocalized to the cytosol, and has alpha-enolase activity. It has beenreported that the monomeric form of this isoform functions as astructural lens protein (tau-crystallin), and the dimeric form as anenolase. In a preferred embodiment of the invention, Eno1 is thetranscript variant 1 of Eno1.

Protein and mRNA sequences of the Homo sapiens enolase 1, (alpha)(ENO1), transcript variant 2, mRNA can be found at GenBank Accession No.NM_(—)001201483 (see www.ncbi.nlm.nih.gov/nuccore/NM_(—)001201483.1,which is incorporated by reference in the version available on the dateof filing the instant application). This variant differs at the 5′ endcompared to variant 1, and initiates translation from an in-framedownstream start codon, resulting in a shorter isoform (MBP-1). Thisisoform is localized to the nucleus, and functions as a transcriptionalrepressor of c-myc protooncogene by binding to its promoter. In certainembodiments of the invention, Eno1 is the transcript variant 2 of Eno1.

Several additional variants of the Eno1 protein have been described, forexample, in the UniProtKB/Swiss-Prot database under Accession No.P06733. Examples of Eno1 protein variants are shown in Table 1 below.

TABLE 1 Eno1 variants. AA res- idue Modification AA modification 2N-acetylserine AA modification 5 N6-acetyllysine AA modification 44Phosphotyrosine AA modification 60 N6-acetyllysine; alternate AAmodification 60 N6-succinyllysine; alternate AA modification 64N6-acetyllysine AA modification 71 N6-acetyllysine AA modification 89N6-acetyllysine; alternate AA modification 89 N6-succinyllysine;alternate AA modification 92 N6-acetyllysine AA modification 126N6-acetyllysine AA modification 193 N6-acetyllysine AA modification 199N6-acetyllysine AA modification 202 N6-acetyllysine AA modification 228N6-acetyllysine; alternate AA modification 228 N6-succinyllysine;alternate AA modification 233 N6-acetyllysine; alternate AA modification233 N6-malonyllysine; alternate AA modification 254 Phosphoserine AAmodification 256 N6-acetyllysine AA modification 263 Phosphoserine AAmodification 272 Phosphoserine AA modification 281 N6-acetyllysine AAmodification 285 N6-acetyllysine AA modification 287 Phosphotyrosine AAmodification 335 N6-acetyllysine AA modification 343 N6-acetyllysine AAmodification 406 N6-acetyllysine AA modification 420 N6-acetyllysine;alternate AA modification 420 N6-malonyllysine; alternate AAmodification 420 N6-succinyllysine; alternate Natural variant 177 N → K.Corresponds to variant rs11544513 [dbSNP| Ensembl]. Natural variant 325P → Q. Corresponds to variant rs11544514 [dbSNP| Ensembl]. Mutagenesis94 M → I: MBP1 protein production. No MBP1 protein production; whenassociated with I-97. Mutagenesis 97 M → I: MBP1 protein production. NoMBP1 protein production; when associated with I-94. Mutagenesis 159Dramatically decreases activity levels Mutagenesis 168 Dramaticallydecreases activity levels Mutagenesis 211 Dramatically decreasesactivity levels Mutagenesis 345 Dramatically decreases activity levelsMutagenesis 384 L → A: Loss of transcriptional repression and cellgrowth inhibition; when associated with A-388. Mutagenesis 388 L → A:Loss of transcriptional repression and cell growth inhibition; whenassociated with A-384. Mutagenesis 396 Dramatically decreases activitylevels

In certain embodiments of the invention, Eno1 is one of the variantslisted in Table 1.

Eno1 Activity

Eno1 is a key glycolytic enzyme that catalyzes the dehydratation of2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in the laststeps of the catabolic glycolytic pathway. Diaz-Ramos et al., 2012, JBiomed Biotechnol. 2012: 156795 and FIG. 33. Enolase enzymes catalysethe dehydration of PGA to PEP in the Emden Mayerhoff-Parnas glycolyticpathway (catabolic direction). In the anabolic pathway (reversereaction) during gluconeogenesis, Eno1 catalyses hydration of PEP toPGA. Accordingly Eno1 is also known as phosphopyruvate hydratase. Metalions are cofactors impairing the increase of enolase activity; henceEno1 is also called metal-activated metalloenzyme. Magnesium is anatural cofactor causing the highest activity and is required for theenzyme to be catalytically active. The relative activation strengthprofile of additional metal ions involved in the enzyme activity appearsin the following order Mg²⁺>Zn²⁺>Mn²⁺>Fe(II)²⁺>Cd²⁺>Co²⁺, Ni²⁺, Sm³⁺,Tb³⁺ and most other divalent metal ions. In reactions catalyzed byenolases, the alpha-proton from a carbon adjacent to a carboxylate groupof PGA, is abstracted, and PGA is conversed to enolate anionintermediate. This intermediate is further processed in a variety ofchemical reactions, including racemization, cycloisomerization andbeta-elimination of either water or ammonia. See Atlas of Genetics andCytogenetics in Oncology and Haematology database,atlasgeneticsoncology.org/Genes/GC_ENO1.html.

Enzymatically active enolase exists in a dimeric (homo- or heterodimers)form and is composed of two subunits facing each other in anantiparallel fashion. The crystal structure of enolase from yeast andhuman has been determined and catalytic mechanisms have been proposed.Diaz-Ramos et al., cited above. The five residues that participate incatalytic activity of this enzyme are highly conserved throughoutevolution. Studies in vitro revealed that mutant enolase enzymes thatdiffer at positions Glu168, Glu211, Lys345, Lys396 or His159,demonstrate dramatically decreased activity levels. An integral andconserved part of enolases are two Mg2+ ions that participate inconformational changes of the active site of enolase and enable bindingof a substrate or its analogues. Atlas of Genetics and Cytogenetics inOncology database, cited above. In certain embodiments, the compositionsof the invention comprise a metal ion cofactor. The metal ion cofactorcan provide increased stability of the Eno1 in the composition and/orincreased activity of the Eno1 in vivo. In one embodiment, the metal ioncofactor is divalent. In one embodiment, the divalent metal ion cofactoris Mg²⁺, Zn²⁺, Mn²⁺, Fe(II)²⁺, Cd²⁺, Co²⁺, or Ni²⁺. In one embodiment,the metal ion cofactor is trivalent, e.g. Sm³⁺ or Tb3⁺.

Eno1 activity may be determined, for example, using the pyruvate kinase(PK)/lactate dehydrogenase (LDH) assay. The reaction for this enolaseassay is shown below.

The rate of reaction of NADH to NAD⁺ conversion may be determined bymeasuring the decrease of fluorescence of NADH, for example by using aPTI Quantamaster 40 spectrophotometer from Photon TechnologyInternational, Inc. (pti-nj.com). Kits for measuring Eno1 activity by acolorimetric pyruvate kinase/lactate dehydrogenase assay are alsocommercially available, for example, from ABCAM (Cambridge, Mass.; Cat.No. ab117994). The ABCAM Eno1 activity assay is further described inExample 5 below.

Eno1 activity may also be determined by measuring the effect of Eno1 onglucose uptake in human skeletal muscle myotubes (HSMM) as described inExample 2.

In certain embodiments, the Eno1 or the fragment thereof has at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400% or 500% of theactivity of a purified endogenous human Eno1 polypeptide. In certainembodiments, the activity of the Eno1, the fragment thereof, and thepurified endogenous human Eno1 polypeptide are determined by thepyruvate kinase/lactate dehydrogenase assay or the HSMM glucose uptakeassay described above.

In certain embodiments, the Eno1 polypeptide in complex with a dendrimeras described herein has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,300%, 400% or 500% of the activity of a purified endogenous Eno1polypeptide that is not in complex with a dendrimer. In certainembodiments, the activity of the Eno1 polypeptide in complex with adendrimer and the activity of the purified endogenous Eno1 polypeptidethat is not in complex with a dendrimer are determined by the pyruvatekinase/lactate dehydrogenase assay or the HSMM glucose uptake assaydescribed above.

In certain embodiments the Eno1 polypeptide in complex with a dendrimerand a targeting peptide as described herein has at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 300%, 400% or 500% of the activity of a purifiedendogenous ENO1 polypeptide that is not in complex with a dendrimer or atargeting peptide. In certain embodiments the activity of the Eno1polypeptide in complex with a dendrimer and a targeting peptide and theactivity of the purified endogenous ENO1 polypeptide that is not incomplex with a dendrimer or a targeting peptide are determined by thepyruvate kinase/lactate dehydrogenase assay or the HSMM glucose uptakeassay described above.

In one embodiment, the Eno1 or the fragment thereof in the compositionof the invention, wherein the composition comprises a metal ion cofactor(e.g., a divalent metal ion cofactor, e.g., Mg²⁺, Zn²⁺, Mn²⁺, Fe(II)²⁺,Cd²⁺, Co²⁺, or Ni²⁺, or a trivalent metal ion cofactor, e.g. Sm³⁺ orTb3⁺) has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%or 500% of the activity of a purified endogenous human Eno1 polypeptide.In certain embodiments, the activity of the Eno1 or the fragment thereofin the composition comprising a metal ion cofactor as described aboveand the activity of the purified endogenous human Eno1 polypeptide aredetermined by the pyruvate kinase/lactate dehydrogenase assay or theHSMM glucose uptake assay described above.

Glucose Flux

The regulation of muscle glucose uptake involves a three-step processconsisting of: (1) delivery of glucose to muscle, (2) transport ofglucose into the muscle by the glucose transporter GLUT4 and (3)phosphorylation of glucose within the muscle by a hexokinase (HK). Thephysiological regulation of muscle glucose uptake requires that glucosetravels from the blood to the interstitium to the intracellular spaceand is then phosphorylated to G6P. Blood glucose concentration, muscleblood flow and recruitment of capillaries to muscle determine glucosemovement from the blood to the interstitium. Plasma membrane GLUT4content controls glucose transport into the cell. Muscle hexokinase (HK)activity, cellular HK compartmentalization and the concentration of theHK inhibitor, G6P, determine the capacity to phosphorylate glucose.These three steps—delivery, transport and phosphorylation ofglucose—comprise glucose flux, and all three steps are important forglucose flux control. However steps downstream of glucosephosphorylation may also affect glucose uptake. For example,acceleration of glycolysis or glycogen synthesis could reduce G6P,increase HK activity, increase the capacity for glucose phosphorylationand potentially stimulate muscle glucose uptake. Wasserman et al., 2010,J Experimental Biology, Vol. 214, pp. 254-262.

The present invention is based, at least in part, on the discovery thatEno1 affects several components of the glucose flux pathway, includingincreasing expression of the glucose transporters GLUT1 and GLUT4 andthe hexokinase HK2, and increasing levels of the glycolysis pathwayintermediates G6P and PEP, thus indicating that Eno1 treatment acts toincrease glucose flux.

The present invention is also based, at least in part, on the discoverythat Eno1 is differentially regulated in muscle cells from normalsubjects and muscle cells from subjects with type 2 diabetes. Theinvention is further based on the surprising discovery that treatment ofmuscle cells with Eno1 increases glucose uptake into the cells andadministration of Eno1 to mice with diet induced obesity normalizesglucose tolerance and insulin response.

Accordingly, the invention provides methods for treatment of elevatedblood glucose typically related to diabetes including at least type 1diabetes, pre-diabetes, type 2 diabetes, and gestational diabetes byadministration of Eno1 to the subject. Further, the invention providesmethods for diagnosing and/or monitoring (e.g., monitoring of diseaseprogression or treatment) and/or prognosing an elevated blood glucosestate, e.g., diabetes, in a mammal. The invention also provides methodsfor treating or for adjusting treatment regimens based on diagnosticinformation relating to the levels of Eno1 in the blood or serum of asubject with elevated blood glucose. The invention further providespanels and kits for practicing the methods of the invention.

The invention also provides methods for increasing glucose flux in asubject comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof. In certainembodiments, the pharmaceutical composition administered to the subjectis any of the pharmaceutical compositions described herein. Theinvention also provides a method of increasing glucose flux in askeletal muscle cell of a subject, the method comprising administeringto the subject a pharmaceutical composition comprising Eno1 or afragment thereof. In certain embodiments, the pharmaceutical compositionadministered to the subject is any of the pharmaceutical compositionsdescribed herein.

The invention also provides a method of increasing glycolytic activityin a skeletal muscle cell of a subject, the method comprisingadministering to the subject a pharmaceutical composition comprisingEno1 or a fragment thereof. In certain embodiments, the pharmaceuticalcomposition administered to the subject is any of the pharmaceuticalcompositions described herein.

The invention also provides a method of increasing mitochondrial freefatty acid oxidation in a skeletal muscle cell of a subject, the methodcomprising administering to the subject a pharmaceutical compositioncomprising Eno1 or a fragment thereof. In certain embodiments, thepharmaceutical composition administered to the subject is any of thepharmaceutical compositions described herein.

“Increasing glucose flux” as used herein is understood as increasing atleast one or more of (1) delivery of glucose to muscle, (2) transport ofglucose into the muscle, and (3) phosphorylation of glucose within themuscle. In particular embodiments, increasing glucose flux includesincreasing glycolytic activity or mitochondrial free fatty acidoxidation in a muscle cell.

IIA. Enolase 2 and Enolase 3

Enolase 2 (Eno2) is also known as gamma enolase, neuronal enolase,neuron-specific enolase (NSE), or HEL-S-279 and is encoded by the ENO2gene. Eno2 is a phosphopyruvate phosphatase, a glycolytic enzyme. Eno2is a homodimer and is found in mature neurons of the central nervoussystem (CNS) and cells of neuronal origin. Neurons under stress ofvarious types release Eno2 into the systemic circulation. Yee, et al.,2012, Invest. Ophthalmol. Vis. Sci. Vol. 53, No. 10, pp. 6389-6392. Thenucleic acid sequence of the ENO2 mRNA and the amino acid sequence ofEno2 are shown in FIGS. 36A and 36B, respectively.

Enolase 3 (Eno3) is also known as beta enolase, muscle enolase,muscle-specific enolase (MSE), or GSD13 and is encoded by the ENO3 gene.Eno3 catalyzes the interconversion of 2-phosphoglycerate andphosphoenolpyruvate. Eno3 is found in adult skeletal muscle cells whereit may play a role in muscle development and regeneration. In adulthuman muscle, over 90% of enolase activity is accounted for by Eno3.Mutations in the gene encoding Eno3 have been associated with glycogenstorage disease. Comi et al., 2001, Ann Neurol. Vol. 50, No. 2, pp.202-207. Three variants of ENO3 mRNA have been identified, variants 1, 2and 3. Variants 1 and 2 encode isoform 1 of the Eno3 protein, andvariant 3 encodes isoform 2 of the Eno3 protein. Variant 3 of the ENO3mRNA differs in the 5′ UTR and lacks two exons in the 5′ coding regioncompared to variant 1. Isoform 2 of the Eno3 protein is shorter thanisoform 1, but has the same N- and C-termini. The nucleic acid sequencesof variants 1, 2 and 3 and amino acid sequences of isoforms 1 and 2 ofEno3 are shown in FIGS. 37A-37E.

Eno2 and/or Eno3 may alternatively also be used in the methods,pharmaceutical compositions, panels, and kits described herein for Eno1.For example, Eno2 and/or Eno3 may be used in methods for treatment ofelevated blood glucose typically related to diabetes including at leasttype 1 diabetes, pre-diabetes, type 2 diabetes, and gestational diabetesby administration of a pharmaceutical composition comprising Eno2 and/orEno3 to the subject. Further, Eno2 and/or Eno3 may be used in methodsfor diagnosing and/or monitoring (e.g., monitoring of diseaseprogression or treatment) and/or prognosing an elevated blood glucosestate, e.g., diabetes, in a mammal. Eno2 and/or Eno3 may also be used inmethods for treating or for adjusting treatment regimens based ondiagnostic information relating to the levels of Eno2 or Eno3 in theblood or serum of a subject with elevated blood glucose, and for panelsand kits for practicing the methods of the invention. The invention alsorelates to pharmaceutical compositions comprising Eno2 and/or Eno3, e.g.for delivery to a muscle cell.

III. DIABETES DIAGNOSIS AND CLASSIFICATION

Diabetes mellitus (DM), often simply referred to as diabetes, is a groupof metabolic diseases in which a person has high blood sugar, eitherbecause the body does not produce enough insulin or because cells do notrespond to the insulin that is produced. This high blood sugar producesthe classical symptoms of polyuria (frequent urination), polydipsia(increased thirst), and polyphagia (increased hunger).

Type 2 diabetes results from insulin resistance, a condition in whichcells fail to use insulin properly, sometimes combined with an absoluteinsulin deficiency. The defective responsiveness of body tissues toinsulin is believed, at least in part, to involve the insulin receptor.However, the specific defects are not known.

In the early stage of type 2 diabetes, the predominant abnormality isreduced insulin sensitivity. At this stage, hyperglycemia can bereversed by a variety of measures and medications that improve insulinsensitivity or reduce glucose production by the liver. Prediabetesindicates a condition that occurs when a person's blood glucose levelsare higher than normal but not high enough for a diagnosis of type 2diabetes.

Type 2 diabetes is due to insufficient insulin production from betacells in the setting of insulin resistance. Insulin resistance, which isthe inability of cells to respond adequately to normal levels ofinsulin, occurs primarily within the muscles, liver, and fat tissue. Inthe liver, insulin normally suppresses glucose release. However in thesetting of insulin resistance, the liver inappropriately releasesglucose into the blood. The proportion of insulin resistance verses betacell dysfunction differs among individuals with some having primarilyinsulin resistance and only a minor defect in insulin secretion andothers with slight insulin resistance and primarily a lack of insulinsecretion.

Other potentially important mechanisms associated with type 2 diabetesand insulin resistance include: increased breakdown of lipids within fatcells, resistance to and lack of incretin, high glucagon levels in theblood, increased retention of salt and water by the kidneys, andinappropriate regulation of metabolism by the central nervous system.However not all people with insulin resistance develop diabetes, sincean impairment of insulin secretion by pancreatic beta cells is alsorequired.

Type 1 diabetes results from the body's failure to produce insulin, andpresently requires treatment with injectable insulin. Type 1 diabetes ischaracterized by loss of the insulin-producing beta cells of the isletsof Langerhans in the pancreas, leading to insulin deficiency. Mostaffected people are otherwise healthy and of a healthy weight when onsetoccurs. Sensitivity and responsiveness to insulin are usually normal,especially in the early stages. However, particularly in late stages,insulin resistance can occur, including insulin resistance due to immunesystem clearance of administered insulin.

A. Diagnostic Criteria

Criteria for diagnosis and classification of diabetes mellitus werepublished by the American Diabetes Association in Diabetes Care,36:S67-74, 2013, incorporated herein by reference, which provides a moredetailed definition of the various types of diabetes. Diagnosticcriteria for diabetes are discussed further below. The referenceclassifies type 1 diabetes or type 2 diabetes as follows:

-   -   I. Type 1 diabetes (β-cell destruction, usually leading to        absolute insulin deficiency)        -   A. Immune mediated        -   B. Idiopathic    -   II. Type 2 diabetes (may range from predominantly insulin        resistance with relative insulin deficiency to a predominantly        secretory defect with insulin resistance)    -   III. Other specific types    -   IV. Gestational diabetes mellitus

Methods for performing diagnostic or assessment methods are providedtherein. The diagnostic criteria for diabetes provided therein are asfollows:

Criteria for the Diagnosis of Diabetes HbA1c ≧6.5%. The test should beperformed in a laboratory using a method that is NationalGlycohemoglobin Standardization Program (NGSP) certified andstandardized to the Diabetes Control and Complications Trial (DCCT)assay.* OR Fasting plasma glucose (FPG) ≧126 mg/dl (7.0 mmol/l). Fastingis defined as no caloric intake for at least 8 h.* OR 2-h plasma glucose≧200 mg/dl (11.1 mmol/l) during an oral glucose tolerance test (OGTT).The test should be performed as described by the World HealthOrganization, using a glucose load containing the equivalent of 75 ganhydrous glucose dissolved in water.* OR In a patient with classicsymptoms of hyperglycemia or hyperglycemic crisis, a random plasmaglucose ≧200 mg/dl (11.1 mmol/l). *In the absence of unequivocalhyperglycemia, criteria 1-3 should be confirmed by repeat testing.

The diagnostic criteria for increased risk of diabetes/pre-diabetesprovided therein are as follows:

Criteria for Increased Risk of Diabetes (Pre-Diabetes)* Fasting PlasmaGlucose (FPG) 100 mg/dl (5.6 mmol/l) to 125 mg/dl (6.9 mmol/l) [ImpairedFasting Glucose - IFG] 2-h Plasma Glucose (PG) in the 75-g oral glucosetolerance test (OGTT) 140 mg/dl (7.8 mmol/l) to 199 mg/dl (11.0 mmol/l)[Impaired Glucose Tolerance - IGT] A1C 5.7-6.4% *For all three tests,risk is continuous, extending below the lower limit of the range andbecoming disproportionately greater at higher ends of the range.

The diagnostic criteria for gestational diabetes provided therein are asfollows:

Screening for and diagnosis of Gestational Diabetes Mellitus (GDM)Perform a 75-g OGTT, with plasma glucose measurement fasting and at 1and 2 h, at 24-28 weeks of gestation in women not previously diagnosedwith overt diabetes. The OGTT should be performed in the morning afteran overnight fast of at least 8 h. The diagnosis of GDM is made when anyof the following plasma glucose values are exceeded: Fasting: ≧92 mg/dl(5.1 mmol/l) 1 h: ≧180 mg/dl (10.0 mmol/l) 2 h: ≧153 mg/dl (8.5 mmol/l)

The blood glucose measurements for the diagnosis and/or monitoring ofelevated blood glucose or diabetes can be cumbersome due to the specifictiming requirements relative to eating, e.g., a fasting blood glucose orthe amount of time required to perform the test, e.g., as with an oralglucose tolerance test. Moreover, the diagnostic criteria explicitlyrequire that in absence of unequivocal hyperglycemia, criteria 1-3should be confirmed by repeat testing. The use of an HbA1c level as adiagnostic indicator can be advantageous as it provides an indication ofblood glucose levels over time, i.e., for about the prior 1-2 months,and does not require special scheduling to perform the test. Similarly,an Eno1 level can be determined without particular schedulingrequirements or food consumption limitations or requirements.

Accordingly, in some aspects the invention relates to a method fordiagnosing the presence of elevated blood glucose in a subject,comprising: (a) contacting a biological sample with a reagent thatselectively binds to Eno1; (b) allowing a complex to form between thereagent and Eno1; (c) detecting the level of the complex, and (d)comparing the level of the complex with a predetermined threshold value,wherein a level of the complex in the sample below the predeterminedthreshold value indicates the subject is suffering from elevated bloodglucose. In certain embodiments, the reagent that selectively binds toEno1 is an anti-Eno1 antibody. In certain embodiments, the antibodycomprises a detectable label.

In some embodiments of the method described above, the step of detectingthe level of the complex further comprises contacting the complex with adetectable secondary antibody and measuring the level of the secondaryantibody. The method may also further comprise detecting the level ofone or more additional indicators of elevated blood glucose. The one ormore additional indicators of blood glucose may be selected from thegroup consisting of HbA1c level, fasting glucose level, fed glucoselevel, and glucose tolerance.

In some embodiments of the aforementioned method, the biological sampleis blood or serum. In some embodiments, the level of the complex isdetected by immunoassay or ELISA. In some embodiments, the presence ofelevated blood glucose in the subject is indicative of a disease orcondition selected from the group consisting of pre-diabetes, type 2diabetes, type 1 diabetes, and gestational diabetes.

B. Secondary Pathologies of Diabetes, Insulin Resistance, and InsulinInsufficiency

Abnormal glucose regulation resulting from diabetes, both type 1 andtype 2, insulin resistance, and insulin insufficiency are associatedwith secondary pathologies, many of which result from poor circulation.Such secondary pathologies include macular degeneration, peripheralneuropathies, ulcers and decrease wound healing, and decreased kidneyfunction. It has been suggested that maintaining glucose levels and/orHbAc1 levels within normal ranges decreases the occurrence of thesesecondary pathologies. It is understood that normalization of bloodglucose, insulin, and HbAc1 levels will reduce the development ofsecondary pathologies by limiting the primary pathology, e.g., impairedglucose tolerance, increased blood glucose. In certain embodiments, Eno1is not used for the treatment of secondary pathologies associated withimpaired glucose tolerance, increased blood glucose, insulin resistance,insulin insufficiency, diabetes, or pre-diabetes. In certainembodiments, Eno1 is used for the treatment of secondary pathologiesassociated with impaired glucose tolerance, increased blood glucose,insulin resistance, insulin insufficiency, diabetes, or pre-diabetes.

IV. DOSAGES AND MODES OF ADMINISTRATION

Techniques and dosages for administration vary depending on the type ofcompound (e.g., protein and/or nucleic acid, alone or complexed with amicroparticle, liposome, or dendrimer) and are well known to thoseskilled in the art or are readily determined.

Therapeutic compounds of the present invention may be administered witha pharmaceutically acceptable diluent, carrier, or excipient, in unitdosage form. Administration may be parenteral, intravenous,subcutaneous, oral, topical, or local. In certain embodiments,administration is not oral. In certain embodiments, administration isnot topical. In certain preferred embodiments, administration issystemic. Administering an agent can be performed by a number of peopleworking in concert. Administering an agent includes, for example,prescribing an agent to be administered to a subject and/or providinginstructions, directly or through another, to take a specific agent,either by self-delivery, e.g., as by oral delivery, subcutaneousdelivery, intravenous delivery through a central line, etc.; or fordelivery by a trained professional, e.g., intravenous delivery,intramuscular delivery, subcutaneous delivery, etc.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous, or parenteral administration; or a polymer orother sustained release vehicle for systemic administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia,Pa.). Formulations for parenteral administration may, for example,contain excipients, sterile water, saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The compound may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids and the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, and the like; andinorganic acid such as hydrochloric acid, hydrobromic acid, sulfuricacid phosphoric acid, and the like. Metal complexes include zinc, iron,and the like.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc). Formulations for oraluse may also be provided as chewable tablets, or as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium.

The dosage and the timing of administering the compound depend onvarious clinical factors including the overall health of the subject andthe severity of the symptoms of disease, e.g., diabetes, pre-diabetes.

A. Formulations for Long Acting Injectable Drugs

Biologics and other agents subject to high rates of first pass clearancemay not be amenable to oral administration and require administration byparenteral routes. However, compliance with treatment regimens forinjectable drugs can be low as subjects are often adverse toself-administering agents by injection, e.g., subcutaneous injection,particularly when the disease does not make the subject feel sick. Otherroutes of administration by injection, e.g., intravenous, intramuscular,typically require administration by a trained professional, makingfrequent administration of the agent inconvenient and often painful.

Formulations have been created to provide sustained delivery ofinjectable agents including, but not limited to, oil-based injections,injectable drug suspensions, injectable microspheres, and injectable insitu systems. Long-acting injectable formulations offer many advantageswhen compared with conventional formulations of the same compounds.These advantages include, at least, the following: a predictabledrug-release profile during a defined period of time following eachinjection; better patient compliance; ease of application; improvedsystemic availability by avoidance of first-pass metabolism; reduceddosing frequency (i.e., fewer injections) without compromising theeffectiveness of the treatment; decreased incidence of side effects; andoverall cost reduction of medical care.

1. Oil-Based Injectable Solutions and Injectable Drug Suspensions.

Conventional long-acting injections consist either of lipophilic drugsin aqueous solvents as suspensions or of lipophilic drugs dissolved invegetable oils. Commercially available oil based injectable drugs forintramuscular administration include, but are not limited to,haloperidol deconate, fluphenazine deconate, testosterone enanthate, andestradiol valerate. Administration frequency for these long-actingformulations is every few weeks or so. In the suspension formulations,the rate-limiting step of drug absorption is the dissolution of drugparticles in the formulation or in the tissue fluid surrounding the drugformulation. Poorly water-soluble salt formations can be used to controlthe dissolution rate of drug particles to prolong the absorption.However, several other factors such as injection site, injection volume,the extent of spreading of the depot at the injection site, and theabsorption and distribution of the oil vehicle per se can affect theoverall pharmacokinetic profile of the drug. Modulation of these factorsto provide the desired drug release profile is within the ability ofthose of skill in the art.

2. Polymer-Based Microspheres and In-Situ Formings.

The development of polymer-based long-acting injectables is one of themost suitable strategies for macromolecules such as peptide and proteindrugs. Commercially available microsphere preparations include, but arenot limited to, leuprolide acetate, triptorelin pamoate, octreotideacetate, lanreotide acetate, risperidone, and naltrexone. Commerciallyavailable in situ forming implants include leuprolide acetate, and insitu forming implants containing paclitaxel and bupivacaine are inclinical trials. These formulations are for intramuscularadministration. Advantages of polymer-based formulations formacromolecules include: in vitro and in vivo stabilization ofmacromolecules, improvement of systemic availability, extension ofbiological half life, enhancement of patient convenience and compliance,and reduction of dosing frequency.

The most crucial factor in the design of injectable microspheres and insitu formings is the choice of an appropriate biodegradable polymer. Therelease of the drug molecule from biodegradable microspheres iscontrolled by diffusion through the polymer matrix and polymerdegradation. The nature of the polymer, such as composition of copolymerratios, polymer crystallinities, glass-transition temperature, andhydrophilicities plays a critical role in the release process. Althoughthe structure, intrinsic polymer properties, core solubility, polymerhydrophilicity, and polymer molecular weight influence the drug-releasekinetics, the possible mechanisms of drug release from microsphere areas follows: initial release from the surface, release through the pores,diffusion through the intact polymer barrier, diffusion through awater-swollen barrier, polymer erosion, and bulk degradation. All thesemechanisms together play a part in the release process. Polymers for usein microsphere and in situ formings include, but are not limited to avariety of biodegradable polymers for controlled drug deliveryintensively studied over the past several decades include polylactides(PLA), polyglycolides (PGA), poly(lactide-co-glycolide) (PLGA),poly(c-caprolactone) (PCL), polyglyconate, polyanhydrides,polyorthoesters, poly(dioxanone), and polyalkylcyanoacrylates. Thermallyinduced gelling systems used in in situ formings show thermo-reversiblesol/gel transitions and are characterized by a lower critical solutiontemperature. They are liquid at room temperature and produce a gel atand above the lower critical solution temperature. In situ solidifyingorganogels are composed of water-insoluble amphiphilic lipids, whichswell in water and form various types of lyotropic liquid crystals.

B. Targeted Drug Delivery

Delivery of drugs to their site of action can increase the therapeuticindex by reducing the amount of drug required to provide the desiredsystemic effect. Drugs can be delivered to the site of action byadministration of the drug to the target tissue using a method orformulation that will limit systemic exposure, e.g., intramuscularinjection, intrasinovial injection, intrathecal injection, intraocularinjection. A number of the sustained delivery formulations discussedabove are for intramuscular administration and provide local delivery tomuscle tissue. Alternatively, targeting moieties can be associated withor linked to therapeutic payloads for administration to the target site.Targeting moieties can include any of a number of moieties that bind tospecific cell types.

1. Targeting Moieties

Certain embodiments of the invention include the use of targetingmoieties include relatively small peptides (e.g., 25 amino acids orless, 20 amino acids or less, 15 amino acids or less, 10 amino acids orless), muscle targeting peptides (MTP) including smooth muscle and/orskeletal muscle targeting peptides, αvβ3 integrin ligands (e.g., RGDpeptides and peptide analogs), αvβ5 integrin ligands, or CD46 ligands asdiscussed above. It is understood that such peptides can include one ormore chemical modifications to permit formation of a complex with Eno1,to modify pharmacokinetic and/or pharmacodynamic properties of thepeptides. In certain embodiments, the targeting moiety can be a smallmolecule, e.g., RGD peptide mimetics. In certain embodiments, thetargeting moiety can include a protein and optionally a fiber proteinfrom an adenovirus 35. In certain embodiments, the viral proteins arepresent on a virus particle. In certain embodiments, the viral proteinsare not present on a viral particle. In certain embodiments, thetargeting moiety can be an antibody, antibody fragment, antibodymimetic, or T-cell receptor.

2. Targeted Complexes

Targeted Eno1 complexes can be administered by a route other thanintramuscular injection (e.g., subcutaneous injection, intravenousinjection) while providing delivery of the Eno1 to muscle. Targetedcomplexes can include one or more targeting moieties attached eitherdirectly or indirectly to Eno1. Formation of the targeted complex doesnot substantially or irreversibly inhibit the activity of Eno1 and itseffect on normalizing blood glucose levels and insulin response. Incertain embodiments, use of a targeted complex can reduce the totalamount of Eno1 required to provide an effective dose. Some exemplary,non-limiting, embodiments of targeted complexes are discussed below.

In certain embodiments, the payload and the targeting moiety are presentin a complex at about a 1:1 molar ratio. In certain embodiments, thetargeting moiety is present in a complex with a molar excess of thepayload (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1; 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1, 15:1, 16:1, 17:1; 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1,25:1, 26:1, 27:1; 28:1, 29:1, 30:1, or more; or any range bracketed byany two values). In certain embodiments, the payload to targeting moietyis about 1:5-1:15; about 1:7-1:13, about 1:8-1:12.

It is understood that the compositions and methods of the inventioninclude the administration of more than one, i.e., a population of,targeting moiety-payload complexes. Therefore, it is understood that thenumber of targeting moieties per payload can represent an average numberof targeting moieties per payload in a population of complexes. Incertain embodiments, at least 70% of the complexes have the selectedmolar ratio of targeting moieties to payload. In certain embodiments, atleast 75% of the complexes have the selected molar ratio of targetingmoieties to payload. In certain embodiments, at least 80% of thecomplexes have the selected molar ratio of targeting moieties topayload. In certain embodiments, at least 85% of the complexes have theselected molar ratio of targeting moieties to payload. In certainembodiments, at least 90% of the complexes have the selected molar ratioof targeting moieties to payload.

a. Linkers

A number of chemical linkers are known in the art and available fromcommercial sources (e.g., Pierce Thermo Fisher Scientific Inc., see,e.g., www.piercenet.com/cat/crosslinking-reagents). Such agents can beused to chemically link, reversibly or irreversibly, one or moretargeting moieties to Eno1. Linkers can also be used to attach targetingmoieties and Eno1 to a structure, e.g., microparticle, dendrimer, ratherthan attaching the targeting moiety directly to Eno1. In certainembodiments, the linker attaching Eno1 to the targeted complex isreversible so that the Eno1 is released from the complex afteradministration, preferably substantially at the muscle.

b. Peptide Bonds

As used herein, targeted complexes can include the translation of Eno1with a peptide targeting moiety. Methods to generate expressionconstructs including an amino acid sequence for targeting Eno1 is wellwithin the ability of those of skill in the art.

c. Liposomes

Liposomal delivery systems are known in the art including formulationsto limit systemic exposure, thereby reducing systemic exposure and offtarget effects. For example, Doxil® is a composition in whichdoxorubicin encapsulated in long-circulating pegylated liposomes thatfurther comprise cholesterol for treatment of certain types of cancer.Various liposomal formulations of amphotericin B including Ambisome®,Abelcet®, and Amphotec® are formulated for intravenous administration inliposomes or a lipid complex containing various phospholipids,cholesterol, and cholesteryl sulfate. Visudine® is verteporfinformulated as a liposome in egg phosphotidyl glycerol and DMPC forintravenous administration. Liposomal formulations are also known forintramuscular injection. Epaxal® is an inactivated hepatitis A virus andInflexal V® is an inactivated hemaglutinine of influenza virus strains Aand B. Both viral preparations are formulated in combinations of DOPCand DOPE. Such liposomes, or other physiologically acceptable liposomes,can be used for the packaging of Eno1 and subsequent surface decorationwith targeting moieties to delivery Eno1 to the muscle. Additionalmoieties to modulate intracellular trafficking of the liposome can alsobe included. Upon uptake of the liposome into the cell, the liposomereleases the Eno1 thereby allowing it to have its therapeutic effect.

d. Dendrimers

Dendrimers can be used as a scaffold for the attachment of multipletargeting moieties with one or more molecules of Eno1. In certainembodiments, the dendrimer is decorated with targeting moieties prior tocoupling with Eno1.

e. Microparticles

Microparticles can be used as a scaffold for the attachment of multipletargeting moieties with one or more molecules of Eno1 either attached toor encapsulated in the microparticle. In certain embodiments, themicroparticle is decorated with targeting moieties prior to couplingwith Eno1.

f. Viral Vectors

Viral tropisms have long been studied and are used to direct viruses tothe cell type of interest. Parker et al., 2013 (Gene Therapy,20:1158-64) have developed an adenovirus serotype 5 capsite with thefiber and peton of serotype 35 to enhance delivery to skeletal and/orsmooth muscle cells. Such viral vectors and other viral vectors can beused for the delivery of Eno1 expression constructs to muscle cells.

C. Dendrimers

Dendrimers can be used in the context of the invention as the backbonefor targeted complexes for the delivery of non-intramuscularlyadministered Eno1 to muscle. Alternatively, dendrimers can be used tomodulate the pharmacokinetic and pharmacodynamic properties ofintramuscularly administered Eno1. In the compositions and methods ofthe invention, dendrimers are understood to be pharmaceuticallyacceptable dendrimers.

Dendrimer-based platforms have achieved attention for use inpharmaceutical applications. Similar to other polymeric carriers,dendrimers can be synthesized to avoid structural toxicity andimmunogenicity. The dendrimer's ability to mimic the size, solubility,and shape of human proteins makes the technology an ideal choice formany therapeutic and diagnostic applications. Being 1-10 nanometers insize enables dendrimers to efficiently diffuse across the vascularendothelium, internalize into cells, and be rapid cleared by thekidneys. This helps to avoid long-term toxicities and reduces the needfor a rapidly degradable platform. The availability of multiple reactivesurface groups enables the dendrimer to carry a higher payload offunctional molecules, enhancing targeted delivery to the site of action,thereby increasing efficacy.

Dendrimers have been produced or are under commercial development forseveral biomedical applications. A topical, polylysine dendrimer-basedmicrobicide, VivaGel™, has been developed by Starpharma. SuperFect® is adendrimer-based material used for gene transfection. Dendrimer baseddiagnostic tools include Gadomer-17, a magnetic resonance imaging (MRI)contrast agent containing a polylysine dendrimer functionalized withgadolinium chelates, and Stratus® CS, a biosensor for cardiac markers torapidly diagnosis heart attacks.

Dendrimers are defined by their core-shell structure, where thedendrimer approximately doubles in size and number of functional surfacegroups with each additional shell (or generation) added to the core.Shells are synthesized by alternating monomer reactions by means wellknown in the art. Specialized dendrimer backbones can be synthesized byvarying the monomer units. The biological properties of the dendrimerare largely influenced by the chemical backbone and surface termination.For a dendrimer to be an appropriate vehicle for drug delivery in vivo,they must be non-toxic, non-immunogenic, and be capable of targeting andreaching specific locations by crossing the appropriate barriers whilebeing stable enough to remain in circulation. The vast majority of thedendrimers synthesized and published in literature are insoluble inphysiological conditions or are incapable of remaining soluble after theaddition of functional molecules and are inappropriate for biologicalapplications. However, several classes of dendrimers have been shown tobe useful scaffolds for biomedical applications; examples includepolyesters, polylysine, and polypropyleneimine (PPI or DAB) dendrimers.

The most widely used dendrimers in biomedical applications arepoly(amidoamine) (PAMAM) dendrimers. The polyamide backbone synthesizedfrom repeating reactions of methyl acrylate and ethylene-diamine helpsthe macromolecule maintain water solubility and minimizesimmunogenicity. PAMAM dendrimers of different generation also are ableto mimic the size and properties of globular proteins readily found inthe body. The amine-terminated surface of full generation PAMAMdendrimers allows for easy surface modification, enabling the platformto carry and solubilize hydrophobic therapeutic molecules, such asmethotrexate, in physiological conditions. PAMAM dendrimers exhibitlittle non-specific toxicity if the surface amines have been neutralizedor appropriately modified (e.g., acylated).

Active targeting uses a molecule, such as targeting moiety, to mediatedelivery of its payload (drug or otherwise) to cells by binding tocell-specific molecules. Targeting moieties, such as those providedherein, frequently bind through receptors highly expressed on targetcells.

The interactions between the targeting ligand and cell-surface receptorallow the therapeutic agent or payload to selectively reach muscle cellsand even be ushered inside via receptor-mediated processes.

The multivalent effect associated with the display of multiple bindingligands on the dendrimer surface enhances the uptake of the dendriticscaffold compared to single ligands. Multivalent interactions, caused bythe simultaneous binding of multiple ligands, allow for the dendrimersto increase the binding avidities of the platform, even when individualligands have low affinities for the targeted receptor. The PAMAMplatform has been successfully used as a scaffold for the attachment ofmultivalent targeting molecules including antibodies, peptides,T-antigens, and folic acid. The targeting ligands anchor the dendrimersto locations where specific receptors are expressed on cell surfaces.Targeted dendrimer-drug conjugates to deliver a higher dose specificallyto targeted cells while avoiding normal cells, thus avoiding thepotential systemic toxicity.

Neutralizing the surface amines of PAMAM dendrimers with acetyl groupsminimizes toxicity and non-specific dendrimer uptake. The acetyl cappingof the dendrimer also allows for increased clearance from the body,minimizing effects from long-term treatment. PEGylation ofamino-terminated PAMAM dendrimers reduces immunogenicity and increasessolubility. PEG terminated dendrimers have an increased half-life theblood stream as compared to the cationic parent material. Hydroxyl andmethyoxyl terminated polyester dendrimers have been shown to be nontoxicin vivo up at concentrations up to 40 mg/kg. The differences intoxicities between cationic and anionic dendrimers have also beenconfirmed in vivo. Using a zebrafish embryo model, carboxyl terminateddendrimer was significantly less toxic than G4 amine-terminateddendrimer. In the same study, surface modification with RGD also reducedtoxicity.

It will be understood that all of the dendrimers described above andherein may be used in the Eno1 compositions of the invention and theirmethods of use.

In certain embodiments, the ratio of the number of dendrimer moleculesto the number of Eno1 molecules in the complex comprising dendrimer andEno1 is between about 1:1 and about 10:1, e.g., about 1:1, about 2:1,about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about9:1, or about 10:1. In one embodiment, the ratio of the number ofdendrimer molecules to the number of Eno1 molecules in the complexcomprising dendrimer and Eno1 is between about 3:1 and 7:1, e.g., 3:1,4:1, 5:1, 6:1, or 7:1. In one embodiment, the ratio of the number ofdendrimer molecules to the number of Eno1 molecules in the complexcomprising dendrimer and Eno1 is between 4:1 and 6:1, e.g., 3:1, 4:1, or5:1. In one embodiment, the ratio of the number of dendrimer moleculesto the number of Eno1 molecules in the complex comprising dendrimer andEno1 is between 3:1 and 5:1, e.g., 3:1, 4:1, or 5:1. In yet anotherembodiment, the ratio of the number of dendrimer molecules to the numberof Eno1 molecules in the complex comprising dendrimer and Eno1 isbetween 4:1 and 5:1. In another embodiment, the ratio of the number ofdendrimer molecules to the number of Eno1 molecules in the complexcomprising dendrimer and Eno1 is between 3:1 and 4:1. In a furtherpreferred embodiment, the ratio of the number of dendrimer molecules tothe number of Eno1 molecules in the complex comprising dendrimer andEno1 is about 5:1.

Optimal ratios of dendrimer to Eno1 in the complex may be tested andselected by assaying the Eno1 activity of the dendrimer/Eno1 complexes(e.g., as compared to uncomplexed Eno1) by using any routine methodsknown in the art, such as, for example, the pyruvate kinase (PK)/lactatedehydrogenase (LDH) assay or any other assays described herein. Optimalratios of dendrimer to Eno1 may also be tested and selected by assessingthe effect of the dendrimer/Eno1 complexes on glucose uptake in an invitro assay, for example, by measuring glucose uptake in human skeletalmuscle myotubes (HSMM) as described herein in Example 2 or any similarassays known in the art. Optimal ratios of dendrimer to Eno1 may also betested and selected by measuring the effect of the dendrimer/Eno1complexes on blood glucose levels in vivo, for example, by measuring theeffect of the dendrimer/Eno1 complex on blood glucose in diabetic mousemodels, as described herein in Examples 7 and 8, or any similar modelsor assays known in the art. Optimal ratios of dendrimer to Eno1 in thecomplex will preferably retain Eno1 activity in vitro and/or in vivo,and/or provide delivery of Eno1 to cells.

It is understood that the compositions and methods of the inventioninclude the administration of more than one, i.e., a population ofdendrimer-Eno1-targeting peptide complexes. Therefore, it is understoodthat the number of dendrimer per Eno1 molecules can represent an averagenumber of dendrimer per Eno1 in a population of complexes. In certainembodiments, at least 70% of the complexes have the selected molar ratioof dendrimer to Eno1. In certain embodiments, at least 75% of thecomplexes have the selected molar ratio of dendrimer to Eno1. In certainembodiments, at least 80% of the complexes have the selected molar ratioof dendrimer to Eno1. In certain embodiments, at least 85% of thecomplexes have the selected molar ratio of dendrimer to Eno1. In certainembodiments, at least 90% of the complexes have the selected molar ratioof dendrimer to Eno1.

In certain embodiments, the ratio of the number of dendrimer moleculesto the number of targeting peptides in the dendrimer/Eno1/targetingpeptide complex is between 1:0.1 and 1:10, between 1:1 and 1:10, between1:1 and 1:5, or between 1:1 and 1:3. In certain embodiments the ratio ofthe number of dendrimer molecules to the number of targeting peptides isabout 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. Ina preferredembodiment, the ratio of the number of dendrimer molecules to the numberof targeting peptides in the dendrimer/Eno1/targeting peptide complex isabout 1:1. In a preferred embodiment, the ratio of the number ofdendrimer molecules to the number of targeting peptides in thedendrimer/Eno1/targeting peptide complex is about 1:2. In a preferredembodiment, the ratio of the number of dendrimer molecules to the numberof targeting peptides in the dendrimer/Eno1/targeting peptide complex isabout 1:3.

In certain embodiments, the ratio of the number of targeting peptides tothe number of dendrimer molecules in the dendrimer/Eno1/targetingpeptide complex is at least 1:1, at least 2:1, at least 3:1, at least4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least9:1 or at least 10:1. In one embodiment, the ratio of the number oftargeting peptides to the number of dendrimer molecules in thedendrimer/Eno1/targeting peptide complex is at least 3:1.

It is understood that the compositions and methods of the inventioninclude the administration of more than one, i.e., a population oftargeting peptide-Eno1-dendrimer complexes. Therefore, it is understoodthat the number of targeting peptides per dendrimer can represent anaverage number of targeting peptides per dendrimer in a population ofcomplexes. In certain embodiments, at least 70% of the complexes havethe selected molar ratio of targeting peptides to dendrimer. In certainembodiments, at least 75% of the complexes have the selected molar ratioof targeting peptides to dendrimer. In certain embodiments, at least 80%of the complexes have the selected molar ratio of targeting peptide todendrimer. In certain embodiments, at least 85% of the complexes havethe selected molar ratio of targeting peptide to dendrimer. In certainembodiments, at least 90% of the complexes have the selected molar ratioof targeting peptide to dendrimer.

Optimal ratios of dendrimer to targeting peptide may be selected bymeasuring the targeting of the dendrimer/Eno1/targeting peptide complexto specific tissues in vivo, for example, by measuring the targeting ofa detectably labeled dendrimer/Eno1/targeting peptide complex in vivo,as described herein in Example 6.

V. DETECTION AND MEASUREMENT OF INDICATORS OF BLOOD GLUCOSE LEVELS ANDCONTROL

Methods for detection and measurement of indicators of elevated bloodglucose and blood glucose control vary depending on the nature of theindicator to be measured. Elevated blood glucose, and thereby loss ofblood glucose level control and severity of diabetes can be measureddirectly, e.g., by determining the amount of glucose in the blood, orindirectly, e.g., by detecting the amount of glycated hemoglobin(HbA1c), a reaction product of hemoglobin and glucose. The inventionfurther provides methods for detecting blood glucose control using Eno1.

The present invention contemplates any suitable means, techniques,and/or procedures for detecting and/or measuring the blood glucose levelindicators of the invention. The skilled artisan will appreciate thatthe methodologies employed to measure the indicators of the inventionwill depend at least on the type of indicator being detected or measured(e.g., glucose, ketones, mRNA, or polypeptide including a glycatedpolypeptide) and the biological sample (e.g., whole blood, serum).Certain biological sample may also require certain specializedtreatments prior to measuring the biomarkers of the invention, e.g., thepreparation of mRNA in the case where an mRNA biomarker, e.g., Eno1mRNA, is being measured.

A. Direct and Indirect Measurement of Blood Glucose and Blood GlucoseControl Using Established Indicators

Blood glucose monitoring is a way of testing the concentration ofglucose in the blood (glycemia) directly at a single point in time.Particularly important in the care of diabetes mellitus, a blood glucosetest is performed by piercing the skin (typically, on the finger) todraw blood, then applying the blood to a chemically active disposable‘test-strip’. Different manufacturers use different technology, but mostsystems measure an electrical characteristic, and use this to determinethe glucose level in the blood. The test is usually referred to ascapillary blood glucose. Commercially available blood glucose monitorsfor periodic or continuous use are known in the art. Glucose monitorsfor periodic detection of blood glucose levels include, but are notlimited to, TRUEResult Blood Glucose Meter (TRUE), ACCU-CHEK GlucoseMeter (ACCU-CHEK), OneTouch Glucose Meter (ONETOUCH), and FreeStyle LiteBlood Glucose (FREESTYLE LITE). It is understood that a directlymeasured normal blood glucose level will vary depending on the amount oftime since food was last consumed with a normal fasting blood glucoselevel being lower than a normal fed blood glucose level. Direct bloodglucose monitoring is also used in glucose tolerance tests to monitorresponse to consumption of a high dose of glucose and the rate ofglucose clearance from the blood.

Glycated hemoglobin (hemoglobin A1c, HbA1c, A1C, Hb1c, HbA1c) is a formof hemoglobin that is measured primarily to identify the average plasmaglucose concentration over prolonged periods of time, i.e., an indirectmeasurement of blood glucose. HbA1c is formed in a non-enzymaticglycation pathway by hemoglobin's exposure to plasma glucose. Whennormal levels of glucose are present, a normal amount of glycatedhemoglobin, measured as a percent of total hemoglobin, or a specificblood concentration, is produced. When blood glucose levels are high,elevated levels of glycated hemoglobin are produced. Glycation is anirreversible reaction. Therefore, the amount of glycated hemoglobinwithin the red cell reflects the average level of glucose to which thecell has been exposed. Measuring glycated hemoglobin assesses theeffectiveness of therapy by monitoring long-term serum glucoseregulation rather than a snapshot image as provided by glucosemonitoring. The HbA1c level is proportional to average blood glucoseconcentration over the previous four weeks to three months. HbA1c levelscan be measured, for example, using high-performance liquidchromatography (HPLC) or immunoassay. Methods for detection andmeasurement of protein analytes are discussed in detail below.

B. Detection of Nucleic Acid Indicators

In certain embodiments, the invention involves the detection of nucleicacid biomarkers, e.g., Eno1 mRNA biomarkers, optionally in combinationwith other indicators of blood glucose, to monitor diabetes and/orglucose control in a subject e.g., direct measurement of blood glucose,ketones, and/or HbA1c.

In various embodiments, the diagnostic/prognostic methods of the presentinvention generally involve the determination of expression levels ofEno1 in a blood sample. Determination of gene expression levels in thepractice of the inventive methods may be performed by any suitablemethod. For example, determination of gene expression levels may beperformed by detecting the expression of mRNA expressed from a gene ofinterest and/or by detecting the expression of a polypeptide encoded bythe gene.

For detecting nucleic acids encoding Eno1, any suitable method can beused, including, but not limited to, Southern blot analysis, northernblot analysis, polymerase chain reaction (PCR) (see, for example, U.S.Pat. Nos. 4,683,195; 4,683,202, and 6,040,166; “PCR Protocols: A Guideto Methods and Applications”, Innis et al. (Eds), 1990, Academic Press:New York), reverse transcriptase PCR (RT-PCR), anchored PCR, competitivePCR (see, for example, U.S. Pat. No. 5,747,251), rapid amplification ofcDNA ends (RACE) (see, for example, “Gene Cloning and Analysis: CurrentInnovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, forexample, EP 01 320 308), one-sided PCR (Ohara et al., Proc. Natl. Acad.Sci., 1989, 86: 5673-5677), in situ hybridization, Taqman-based assays(Holland et al., Proc. Natl. Acad. Sci., 1991, 88: 7276-7280),differential display (see, for example, Liang et al., Nucl. Acid. Res.,1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleicacid sequence based amplification (NASBA) and other transcription basedamplification systems (see, for example, U.S. Pat. Nos. 5,409,818 and5,554,527), Qbeta Replicase, Strand Displacement Amplification (SDA),Repair Chain Reaction (RCR), nuclease protection assays,subtraction-based methods, Rapid-Scan®, etc.

In other embodiments, gene expression levels of Eno1 may be determinedby amplifying complementary DNA (cDNA) or complementary RNA (cRNA)produced from mRNA and analyzing it using a microarray. A number ofdifferent array configurations and methods of their production are knownto those skilled in the art (see, for example, U.S. Pat. Nos. 5,445,934;5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756;5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695;5,624,711; 5,658,734; and 5,700,637). Microarray technology allows forthe measurement of the steady-state mRNA level of a large number ofgenes simultaneously. Microarrays currently in wide use include cDNAarrays and oligonucleotide arrays. Analyses using microarrays aregenerally based on measurements of the intensity of the signal receivedfrom a labeled probe used to detect a cDNA sequence from the sample thathybridizes to a nucleic acid probe immobilized at a known location onthe microarray (see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114;6,218,122; and 6,271,002). Array-based gene expression methods are knownin the art and have been described in numerous scientific publicationsas well as in patents (see, for example, M. Schena et al., Science,1995, 270: 467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996,93: 10614-10619; J. J. Chen et al., Genomics, 1998, 51: 313-324; U.S.Pat. Nos. 5,143,854; 5,445,934; 5,807,522; 5,837,832; 6,040,138;6,045,996; 6,284,460; and 6,607,885).

In one particular embodiment, the invention comprises a method foridentification of a subject suffering from abnormal blood glucose byamplifying and detecting nucleic acids corresponding to Eno1, optionallyin combination with one or more additional indicators of elevated bloodglucose.

Nucleic acid used as a template for amplification can be isolated fromcells contained in the biological sample, according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary cDNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to any of the Eno1 nucleotide sequences identified hereinare contacted with the isolated nucleic acid under conditions thatpermit selective hybridization. Once hybridized, the nucleic acid:primercomplex is contacted with one or more enzymes that facilitatetemplate-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced. Next, theamplification product is detected. In certain applications, thedetection may be performed by visual means. Alternatively, the detectionmay involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (AFFYMAX technology; Bellus, 1994). Following detection,one may compare the results seen in a given patient with a statisticallysignificant reference group of normal patients and patients withelevated blood glucose, e.g., patients with pre-diabetes, type 2diabetes, gestational diabetes, or type 1 diabetes. In this way, it ispossible to correlate the amount of nucleic acid detected with variousclinical states.

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty, preferably fifteen to twenty nucleotides in length,but longer sequences may be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

A number of template dependent processes are available to amplify thenucleic acid sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in its entirety.

In PCR, two primer sequences are prepared which are complementary toregions on opposite complementary strands of the target nucleic acidsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe target nucleic acid sequence is present in a sample, the primerswill bind to the target nucleic acid and the polymerase will cause theprimers to be extended along the target nucleic acid sequence by addingon nucleotides. By raising and lowering the temperature of the reactionmixture, the extended primers will dissociate from the target nucleicacid to form reaction products, excess primers will bind to the targetnucleic acid and to the reaction products and the process is repeated.

A reverse transcriptase PCR amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable DNA polymerases. These methods are described in WO 90/07641filed Dec. 21, 1990. Polymerase chain reaction methodologies are wellknown in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirely. In LCR, two complementary probe pairs areprepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes amethod similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, alsomay be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA which has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence which may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]-triphosphates in one strand of arestriction site also may be useful in the amplification of nucleicacids in the present invention. Walker et al. (1992), incorporatedherein by reference in its entirety.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases may be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencesalso may be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still other amplification methods described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR like, template and enzyme dependentsynthesis. The primers may be modified by labeling with a capture moiety(e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labeled probes are added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labeled probe signals thepresence of the target sequence.

Other contemplated nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR. Kwoh et al. (1989);Gingeras et al., PCT Application WO 88/10315, incorporated herein byreference in their entirety. In NASBA, the nucleic acids may be preparedfor amplification by standard phenol/chloroform extraction, heatdenaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by a polymerase such as T7 or SP6. In anisothermal cyclic reaction, the RNA's are reverse transcribed intodouble stranded DNA, and transcribed once against with a polymerase suchas T7 or SP6. The resulting products, whether truncated or complete,indicate target specific sequences.

Davey et al., European Application No. 329 822 (incorporated herein byreference in its entirely) disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H(RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase 1), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence may be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies may thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification may be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence may be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” and “one-sidedPCR™.” Frohman (1990) and Ohara et al. (1989), each herein incorporatedby reference in their entirety.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, alsomay be used in the amplification step of the present invention. Wu etal. (1989), incorporated herein by reference in its entirety.

Oligonucleotide probes or primers of the present invention may be of anysuitable length, depending on the particular assay format and theparticular needs and targeted sequences employed. In a preferredembodiment, the oligonucleotide probes or primers are at least 10nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ),preferably at least 15 nucleotides in length (15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ) and they may beadapted to be especially suited for a chosen nucleic acid amplificationsystem and/or hybridization system used. Longer probes and primers arealso within the scope of the present invention as well known in the art.Primers having more than 30, more than 40, more than 50 nucleotides andprobes having more than 100, more than 200, more than 300, more than 500more than 800 and more than 1000 nucleotides in length are also coveredby the present invention. Of course, longer primers have thedisadvantage of being more expensive and thus, primers having between 15and 30 nucleotides in length are usually designed and used in the art.As well known in the art, probes ranging from 10 to more than 2000nucleotides in length can be used in the methods of the presentinvention. As for the % of identity described above, non-specificallydescribed sizes of probes and primers (e.g., 16, 17, 31, 24, 39, 350,450, 550, 900, 1240 nucleotides, . . . ) are also within the scope ofthe present invention. In one embodiment, the oligonucleotide probes orprimers of the present invention specifically hybridize with an Eno1 RNA(or its complementary sequence) or an Eno1 mRNA. More preferably, theEno1 primers and probes are chosen to detect an Eno1 RNA which isassociated with elevated blood glucose or abnormal blood glucoseregulation related to, e.g., pre-diabetes, type 2 diabetes, type 1diabetes, or gestational diabetes.

In other embodiments, the detection means can utilize a hybridizationtechnique, e.g., where a specific primer or probe is selected to annealto a target biomarker of interest, e.g., Eno1, and thereafter detectionof selective hybridization is made. As commonly known in the art, theoligonucleotide probes and primers can be designed by taking intoconsideration the melting point of hybridization thereof with itstargeted sequence (see below and in Sambrook et al., 1989, MolecularCloning—A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel etal., 1994, in Current Protocols in Molecular Biology, John Wiley & SonsInc., N.Y.).

To enable hybridization to occur under the assay conditions of thepresent invention, oligonucleotide primers and probes should comprise anoligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%,74% or more), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more) and more preferablyat least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%)identity to a portion of an Eno1 polynucleotide. Probes and primers ofthe present invention are those that hybridize under stringenthybridization conditions and those that hybridize to Eno1 homologs underat least moderately stringent conditions. In certain embodiments probesand primers of the present invention have complete sequence identity toEno1 gene sequences (e.g., cDNA or mRNA). It should be understood thatother probes and primers could be easily designed and used in thepresent invention based on the Eno1 sequences disclosed herein by usingmethods of computer alignment and sequence analysis known in the art(cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited byCold Spring Harbor Laboratory, 2000).

C. Detection of Polypeptide Indicators of Blood Glucose of Blood GlucoseControl

The present invention contemplates any suitable method for detectingpolypeptide indicators of blood glucose including Eno1 and HbA1c. Incertain embodiments, the detection method is an immunodetection methodinvolving an antibody that specifically binds to one or more of Eno1 andhemoglobin, especially specifically to glycated hemoglobin. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Nakamura et al. (1987), which isincorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein or peptide indicator of elevated bloodglucose, and contacting the sample with an antibody in accordance withthe present invention, as the case may be, under conditions effective toallow the formation of immunocomplexes.

The immunobinding methods include methods for detecting or quantifyingthe amount of a reactive component in a sample, which methods requirethe detection or quantitation of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containinga protein or peptide indicator of elevated blood glucose, and contactthe sample with an antibody, and then detect or quantify the amount ofimmune complexes formed under the specific conditions.

In terms of detection of an indicator of blood glucose, the biologicalsample analyzed may be any sample that is suspected of containing aprotein or peptide indicator of blood glucose, such as, Eno1 or HbA1c.The biological sample may be, for example, blood, in the case of HbA1c,or blood or serum in the case of Eno1.

Contacting the chosen biological sample with the antibody (e.g., as adetection reagent that binds Eno1, HbA1c, or hemoglobin in a biologicalsample) under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes).Generally, complex formation is a matter of simply adding thecomposition to the biological sample and incubating the mixture for aperiod of time long enough for the antibodies to form immune complexeswith, i.e., to bind to, any antigens present. After this time, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or western blot, is generally washed to remove any non-specificallybound antibody species, allowing only those antibodies specificallybound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

The antibody (e.g., anti-Eno1 antibody, anti-hemoglobin or anti-glycatedhemoglobin antibody) employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined.

Alternatively, the first added component that becomes bound within theprimary immune complexes may be detected by means of a second bindingligand that has binding affinity for the bound antibody. In these cases,the second binding ligand may be linked to a detectable label. Thesecond binding ligand is itself often an antibody, which may thus betermed a “secondary” antibody. The primary immune complexes arecontacted with the labeled, secondary binding ligand, or antibody, underconditions effective and for a period of time sufficient to allow theformation of secondary immune complexes. The secondary immune complexesare then generally washed to remove any non-specifically bound labeledsecondary antibodies or ligands, and the remaining label in thesecondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the encoded protein, peptide or correspondingantibody is used to form secondary immune complexes, as described above.After washing, the secondary immune complexes are contacted with a thirdbinding ligand or antibody that has binding affinity for the secondantibody, again under conditions effective and for a period of timesufficient to allow the formation of immune complexes (tertiary immunecomplexes). The third ligand or antibody is linked to a detectablelabel, allowing detection of the tertiary immune complexes thus formed.This system may provide for signal amplification if this is desired.

The immunodetection methods of the present invention have evidentutility in the diagnosis of conditions such as elevated blood glucose,loss of blood glucose control, and diabetes. Here, a biological orclinical sample suspected of containing either the encoded protein orglycated peptide is used. However, these embodiments also haveapplications to non-clinical samples, such as in the tittering ofantigen or antibody samples, in the selection of hybridomas, and thelike.

The present invention, in particular, contemplates the use of ELISAs asa type of immunodetection assay. It is contemplated that the biomarkerproteins or peptides of the invention will find utility as immunogens inELISA assays in diagnosis and prognostic monitoring abnormal bloodglucose and diabetes. Immunoassays, in their most simple and directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) andradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections can be useful. However, it will be readilyappreciated that detection is not limited to such techniques, andwestern blotting, dot blotting, and the like also may be used.

In one exemplary ELISA, antibodies binding to the protein indicators ofthe invention are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing an indicator of blood glucoselevels, such as a blood or serum sample, is added to the wells. Afterbinding and washing to remove non-specifically bound immunecomplexes,the bound antigen may be detected. Detection is generally achieved bythe addition of a second antibody specific for the indicator protein,that is linked to a detectable label. This type of ELISA is a simple“sandwich ELISA.” Detection also may be achieved by the addition of asecond antibody, followed by the addition of a third antibody that hasbinding affinity for the second antibody, with the third antibody beinglinked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theblood glucose indicator proteins are immobilized onto the well surfaceand then contacted with specific antibodies for binding the indicators.After binding and washing to remove non-specifically boundimmunecomplexes, the bound antigen is detected. Where the initialantibodies are linked to a detectable label, the immunecomplexes may bedetected directly. Again, the immunocomplexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.These are described as follows.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein, and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is customary to use a secondary or tertiary detectionmeans rather than a direct procedure. Thus, after binding of a proteinor antibody to the well, coating with a non-reactive material to reducebackground, and washing to remove unbound material, the immobilizingsurface is contacted with the control biological sample, e.g., blood orserum from a subject with normal blood glucose and/or sufficient bloodglucose control to be tested under conditions effective to allowimmunecomplex (antigen/antibody) formation. Detection of theimmunocomplex then requires a labeled secondary binding ligand orantibody, or a secondary binding ligand or antibody in conjunction witha labeled tertiary antibody or third binding ligand.

The phrase “under conditions effective to allow immunecomplex(antigen/antibody) formation” means that the conditions preferablyinclude diluting the antigens and antibodies with solutions such as BSA,bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween.These added agents also tend to assist in the reduction of nonspecificbackground.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to about 4 hours,at temperatures preferably on the order of 25 to 27° C., or may beovernight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween or boratebuffer. Following the formation of specific immunecomplexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immunecomplexes may bedetermined.

To provide a detecting means, the second or third antibody has anassociated label to allow detection. Preferably, the label is an enzymethat generates color development upon incubating with an appropriatechromogenic substrate. Thus, for example, the first or secondimmunecomplex is contacted and incubated with a urease, glucose oxidase,alkaline phosphatase or hydrogen peroxidase-conjugated antibody for aperiod of time and under conditions that favor the development offurther immunecomplex formation (e.g., incubation for 2 h at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple. Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

The protein biomarkers/indicators of the invention (e.g., Eno1, HbA1c)can also be measured, quantitated, detected, and otherwise analyzedusing protein mass spectrometry methods and instrumentation. Proteinmass spectrometry refers to the application of mass spectrometry to thestudy of proteins. Although not intending to be limiting, two approachesare typically used for characterizing proteins using mass spectrometry.In the first, intact proteins are ionized and then introduced to a massanalyzer. This approach is referred to as “top-down” strategy of proteinanalysis. The two primary methods for ionization of whole proteins areelectrospray ionization (ESI) and matrix-assisted laserdesorption/ionization (MALDI). In the second approach, proteins areenzymatically digested into smaller peptides using a protease such astrypsin. Subsequently these peptides are introduced into the massspectrometer and identified by peptide mass fingerprinting or tandemmass spectrometry. Hence, this latter approach (also called “bottom-up”proteomics) uses identification at the peptide level to infer theexistence of proteins.

Whole protein mass analysis of the biomarkers of the invention can beconducted using time-of-flight (TOF) MS, or Fourier transform ioncyclotron resonance (FT-ICR). These two types of instruments are usefulbecause of their wide mass range, and in the case of FT-ICR, its highmass accuracy. The most widely used instruments for peptide massanalysis are the MALDI time-of-flight instruments as they permit theacquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF canbe analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flightand the quadrupole ion trap also find use in this application.

Protein indicators can also be measured in complex mixtures of proteinsand molecules that co-exist in a biological medium or sample, however,fractionation of the sample may be required and is contemplated herein.It will be appreciated that ionization of complex mixtures of proteinscan result in situation where the more abundant proteins have a tendencyto “drown” or suppress signals from less abundant proteins in the samesample. In addition, the mass spectrum from a complex mixture can bedifficult to interpret because of the overwhelming number of mixturecomponents. Fractionation can be used to first separate any complexmixture of proteins prior to mass spectrometry analysis. Two methods arewidely used to fractionate proteins, or their peptide products from anenzymatic digestion. The first method fractionates whole proteins and iscalled two-dimensional gel electrophoresis. The second method, highperformance liquid chromatography (LC or HPLC) is used to fractionatepeptides after enzymatic digestion. In some situations, it may bedesirable to combine both of these techniques. Any other suitablemethods known in the art for fractionating protein mixtures are alsocontemplated herein.

Gel spots identified on a 2D Gel are usually attributable to oneprotein. If the identity of the protein is desired, usually the methodof in-gel digestion is applied, where the protein spot of interest isexcised, and digested proteolytically. The peptide masses resulting fromthe digestion can be determined by mass spectrometry using peptide massfingerprinting. If this information does not allow unequivocalidentification of the protein, its peptides can be subject to tandemmass spectrometry for de novo sequencing.

Characterization of protein mixtures using HPLC/MS may also be referredto in the art as “shotgun proteomics” and MuDPIT (Multi-DimensionalProtein Identification Technology). A peptide mixture that results fromdigestion of a protein mixture is fractionated by one or two steps ofliquid chromatography (LC). The eluent from the chromatography stage canbe either directly introduced to the mass spectrometer throughelectrospray ionization, or laid down on a series of small spots forlater mass analysis using MALDI.

Protein indicators (e.g., Eno1 or Hb1Ac) can be identified using MSusing a variety of techniques, all of which are contemplated herein.Peptide mass fingerprinting uses the masses of proteolytic peptides asinput to a search of a database of predicted masses that would arisefrom digestion of a list of known proteins. If a protein sequence in thereference list gives rise to a significant number of predicted massesthat match the experimental values, there is some evidence that thisprotein was present in the original sample. It will be furtherappreciated that the development of methods and instrumentation forautomated, data-dependent electrospray ionization (ESI) tandem massspectrometry (MS/MS) in conjunction with microcapillary liquidchromatography (LC) and database searching has significantly increasedthe sensitivity and speed of the identification of gel-separatedproteins. Microcapillary LC-MS/MS has been used successfully for thelarge-scale identification of individual proteins directly from mixtureswithout gel electrophoretic separation (Link et al., 1999; Opitek etal., 1997).

Several recent methods allow for the quantitation of proteins by massspectrometry. For example, stable (e.g., non-radioactive) heavierisotopes of carbon (13C) or nitrogen (15N) can be incorporated into onesample while the other one can be labeled with corresponding lightisotopes (e.g. 12C and 14N). The two samples are mixed before theanalysis. Peptides derived from the different samples can bedistinguished due to their mass difference. The ratio of their peakintensities corresponds to the relative abundance ratio of the peptides(and proteins). The most popular methods for isotope labeling are SILAC(stable isotope labeling by amino acids in cell culture),trypsin-catalyzed 180 labeling, ICAT (isotope coded affinity tagging),iTRAQ (isobaric tags for relative and absolute quantitation).“Semi-quantitative” mass spectrometry can be performed without labelingof samples. Typically, this is done with MALDI analysis (in linearmode). The peak intensity, or the peak area, from individual molecules(typically proteins) is here correlated to the amount of protein in thesample. However, the individual signal depends on the primary structureof the protein, on the complexity of the sample, and on the settings ofthe instrument. Other types of “label-free” quantitative massspectrometry, uses the spectral counts (or peptide counts) of digestedproteins as a means for determining relative protein amounts.

In one embodiment, any one or more of the protein indicators (e.g.,Eno1, HbA1c) can be identified and quantified from a complex biologicalsample using mass spectroscopy in accordance with the followingexemplary method, which is not intended to limit the invention or theuse of other mass spectrometry-based methods.

In the first step of this embodiment, (A) a biological sample, e.g., abiological sample suspected of having increased blood glucose, whichcomprises a complex mixture of protein (including at least one indicatorof interest) is fragmented and labeled with a stable isotope X. (B)Next, a known amount of an internal standard is added to the biologicalsample, wherein the internal standard is prepared by fragmenting astandard protein that is identical to the at least one target biomarkerof interest, and labeled with a stable isotope Y. (C) This sampleobtained is then introduced in an LC-MS/MS device, and multiple reactionmonitoring (MRM) analysis is performed using MRM transitions selectedfor the internal standard to obtain an MRM chromatogram. (D) The MRMchromatogram is then viewed to identify a target peptide biomarkerderived from the biological sample that shows the same retention time asa peptide derived from the internal standard (an internal standardpeptide), and quantifying the target protein indicator in the testsample by comparing the peak area of the internal standard peptide withthe peak area of the target peptide indicator.

Any suitable biological sample may be used as a starting point forLC-MS/MS/MRM analysis, including biological samples derived blood,urine, saliva, hair, cells, cell tissues, biopsy materials, and treatedproducts thereof; and protein-containing samples prepared by generecombination techniques. Preferred embodiments of the invention includethe use of blood or serum samples.

Each of the above steps (A) to (D) is described further below.

Step (A) (Fragmentation and Labeling). In step (A), the target proteinindicator is fragmented to a collection of peptides, which issubsequently labeled with a stable isotope X. To fragment the targetprotein, for example, methods of digesting the target protein with aproteolytic enzyme (protease) such as trypsin, and chemical cleavagemethods, such as a method using cyanogen bromide, can be used. Digestionby protease is preferable. It is known that a given mole quantity ofprotein produces the same mole quantity for each tryptic peptidecleavage product if the proteolytic digest is allowed to proceed tocompletion. Thus, determining the mole quantity of tryptic peptide to agiven protein allows determination of the mole quantity of the originalprotein in the sample. Absolute quantification of the target protein canbe accomplished by determining the absolute amount of the targetprotein-derived peptides contained in the protease digestion (collectionof peptides). Accordingly, in order to allow the proteolytic digest toproceed to completion, reduction and alkylation treatments arepreferably performed before protease digestion with trypsin to reduceand alkylate the disulfide bonds contained in the target protein.

Subsequently, the obtained digest (collection of peptides, comprisingpeptides of the target biomarker in the biological sample) is subjectedto labeling with a stable isotope X. Examples of stable isotopes Xinclude 1H and 2H for hydrogen atoms, 12C and 13C for carbon atoms, and14N and 15N for nitrogen atoms. Any isotope can be suitably selectedtherefrom. Labeling by a stable isotope X can be performed by reactingthe digest (collection of peptides) with a reagent containing the stableisotope. Preferable examples of such reagents that are commerciallyavailable include mTRAQ® (produced by Applied Biosystems), which is anamine-specific stable isotope reagent kit. mTRAQ® is composed of 2 or 3types of reagents (mTRAQ®-light and mTRAQ®-heavy; or mTRAQ®-D0,mTRAQ®-D4, and mTRAQ®-D8) that have a constant mass difference therebetween as a result of isotope-labeling, and that are bound to theN-terminus of a peptide or the primary amine of a lysine residue.

Step (B) (Addition of the Internal Standard). In step (B), a knownamount of an internal standard is added to the sample obtained in step(A). The internal standard used herein is a digest (collection ofpeptides) obtained by fragmenting a protein (standard protein)consisting of the same amino acid sequence as the target protein (targetbiomarker) to be measured, and labeling the obtained digest (collectionof peptides) with a stable isotope Y. The fragmentation treatment can beperformed in the same manner as above for the target protein. Labelingwith a stable isotope Y can also be performed in the same manner asabove for the target protein. However, the stable isotope Y used hereinmust be an isotope that has a mass different from that of the stableisotope X used for labeling the target protein digest. For example, inthe case of using the aforementioned mTRAQ (registered trademark)(produced by Applied Bio systems), when mTRAQ-light is used to label atarget protein digest, mTRAQ-heavy should be used to label a standardprotein digest.

Step (C) (LC-MS/MS and MRM Analysis). In step (C), the sample obtainedin step (B) is first placed in an LC-MS/MS device, and then multiplereaction monitoring (MRM) analysis is performed using MRM transitionsselected for the internal standard. By LC (liquid chromatography) usingthe LC-MS/MS device, the sample (collection of peptides labeled with astable isotope) obtained in step (B) is separated first byone-dimensional or multi-dimensional high-performance liquidchromatography. Specific examples of such liquid chromatography includecation exchange chromatography, in which separation is conducted byutilizing electric charge difference between peptides; andreversed-phase chromatography, in which separation is conducted byutilizing hydrophobicity difference between peptides. Both of thesemethods may be used in combination.

Subsequently, each of the separated peptides is subjected to tandem massspectrometry by using a tandem mass spectrometer (MS/MS spectrometer)comprising two mass spectrometers connected in series. The use of such amass spectrometer enables the detection of several fmol levels of atarget protein. Furthermore, MS/MS analysis enables the analysis ofinternal sequence information on peptides, thus enabling identificationwithout false positives. Other types of MS analyzers may also be used,including magnetic sector mass spectrometers (Sector MS), quadrupolemass spectrometers (QMS), time-of-flight mass spectrometers (TOFMS), andFourier transform ion cyclotron resonance mass spectrometers (FT-ICRMS),and combinations of these analyzers.

Subsequently, the obtained data are put through a search engine toperform a spectral assignment and to list the peptides experimentallydetected for each protein. The detected peptides are preferably groupedfor each protein, and preferably at least three fragments having an m/zvalue larger than that of the precursor ion and at least three fragmentswith an m/z value of, preferably, 500 or more are selected from eachMS/MS spectrum in descending order of signal strength on the spectrum.From these, two or more fragments are selected in descending order ofstrength, and the average of the strength is defined as the expectedsensitivity of the MRR transitions. When a plurality of peptides isdetected from one protein, at least two peptides with the highestsensitivity are selected as standard peptides using the expectedsensitivity as an index.

Step (D) (Quantification of the Target Protein in the Test Sample). Step(D) comprises identifying, in the MRM chromatogram detected in step (C),a peptide derived from the target protein (a target biomarker ofinterest) that shows the same retention time as a peptide derived fromthe internal standard (an internal standard peptide), and quantifyingthe target protein in the test sample by comparing the peak area of theinternal standard peptide with the peak area of the target peptide. Thetarget protein can be quantified by utilizing a calibration curve of thestandard protein prepared beforehand.

The calibration curve can be prepared by the following method. First, arecombinant protein consisting of an amino acid sequence that isidentical to that of the target biomarker protein is digested with aprotease such as trypsin, as described above. Subsequently,precursor-fragment transition selection standards (PFTS) of a knownconcentration are individually labeled with two different types ofstable isotopes (i.e., one is labeled with a stable isomer used to labelan internal standard peptide (labeled with IS), whereas the other islabeled with a stable isomer used to label a target peptide (labeledwith T). A plurality of samples are produced by blending a certainamount of the IS-labeled PTFS with various concentrations of theT-labeled PTFS. These samples are placed in the aforementioned LC-MS/MSdevice to perform MRM analysis. The area ratio of the T-labeled PTFS tothe IS-labeled PTFS (T-labeled PTFS/IS-labeled PTFS) on the obtained MRMchromatogram is plotted against the amount of the T-labeled PTFS toprepare a calibration curve. The absolute amount of the target proteincontained in the test sample can be calculated by reference to thecalibration curve.

D. Antibodies and Labels (e.g., Fluorescent Moieties and Dyes)

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof the biomolecules of the invention, e.g., Eno1 alone or in combinationwith at least one other indicator of blood glucose and blood glucosecontrol, e.g., HbA1c, ketones, or direct measurement of blood glucose.One skilled in the art will recognize that many strategies can be usedfor labeling target molecules to enable their detection ordiscrimination in a mixture of particles (e.g., labeled anti-Eno1antibody or labeled secondary antibody, or labeled oligonucleotide probethat specifically hybridizes to Eno1 mRNA). The labels may be attachedby any known means, including methods that utilize non-specific orspecific interactions of label and target. Labels may provide adetectable signal or affect the mobility of the particle in an electricfield. In addition, labeling can be accomplished directly or throughbinding partners.

In some embodiments, the label comprises a binding partner that binds tothe indicator of interest, where the binding partner is attached to afluorescent moiety. The compositions and methods of the invention mayutilize highly fluorescent moieties, e.g., a moiety capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, wherein the laser is focused ona spot not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. Moieties suitable for thecompositions and methods of the invention are described in more detailbelow.

In some embodiments, the invention provides a label for detecting abiological molecule comprising a binding partner for the biologicalmolecule that is attached to a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the moiety comprises a plurality offluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or3 to 10 fluorescent entities. In some embodiments, the moiety comprisesabout 2 to 4 fluorescent entities. In some embodiments, the biologicalmolecule is a protein or a small molecule. In some embodiments, thebiological molecule is a protein. The fluorescent entities can befluorescent dye molecules. In some embodiments, the fluorescent dyemolecules comprise at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the dye molecules are Alexa Fluor molecules selected fromthe group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dyemolecules are Alexa Fluor molecules selected from the group consistingof Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700.In some embodiments, the dye molecules are Alexa Fluor 647 dyemolecules. In some embodiments, the dye molecules comprise a first typeand a second type of dye molecules, e.g., two different Alexa Fluormolecules, e.g., where the first type and second type of dye moleculeshave different emission spectra. The ratio of the number of first typeto second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1to 1, 1 to 2, 1 to 3 or 1 to 4. The binding partner can be, e.g., anantibody.

In some embodiments, the invention provides a label for the detection ofa biological indicators of the invention, wherein the label comprises abinding partner for the indicator and a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the fluorescent moiety comprises afluorescent molecule. In some embodiments, the fluorescent moietycomprises a plurality of fluorescent molecules, e.g., about 2 to 10, 2to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, or 3 to 6 fluorescent molecules.In some embodiments, the label comprises about 2 to 4 fluorescentmolecules. In some embodiments, the fluorescent dye molecules compriseat least one substituted indolium ring system in which the substituenton the 3-carbon of the indolium ring contains a chemically reactivegroup or a conjugated substance. In some embodiments, the fluorescentmolecules are selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. Insome embodiments, the fluorescent molecules are selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or AlexaFluor 700. In some embodiments, the fluorescent molecules are AlexaFluor 647 molecules. In some embodiments, the binding partner comprisesan antibody. In some embodiments, the antibody is a monoclonal antibody.In other embodiments, the antibody is a polyclonal antibody.

In various embodiments, the binding partner for detecting an indicatorof interest, e.g., Eno1 or HbA1c, is an antibody or antigen-bindingfragment thereof. The term “antibody,” as used herein, is a broad termand is used in its ordinary sense, including, without limitation, torefer to naturally occurring antibodies as well as non-naturallyoccurring antibodies, including, for example, single chain antibodies,chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof. An “antigen-binding fragment” of anantibody refers to the part of the antibody that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. It will be appreciated that the choice of epitope or region ofthe molecule to which the antibody is raised will determine itsspecificity, e.g., for various forms of the molecule, if present, or fortotal (e.g., all, or substantially all of the molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc.,Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA;Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049USA; BiosPacific, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In general, antibodies can be produced by cell culture techniques,including the generation of monoclonal antibodies as described herein,or via transfection of antibody genes into suitable bacterial ormammalian cell hosts, in order to allow for the production ofrecombinant antibodies.

Monoclonal antibodies may be prepared using hybridoma methods, such asthe technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976),and improvements thereto. These methods involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding antibodies employed in the disclosed methods may be isolatedand sequenced using conventional procedures. Recombinant antibodies,antibody fragments, and/or fusions thereof, can be expressed in vitro orin prokaryotic cells (e.g. bacteria) or eukaryotic cells (e.g. yeast,insect or mammalian cells) and further purified as necessary using wellknown methods.

More particularly, monoclonal antibodies (MAbs) may be readily preparedthrough use of well-known techniques, such as those exemplified in U.S.Pat. No. 4,196,265, incorporated herein by reference. Typically, thistechnique involves immunizing a suitable animal with a selectedimmunogen composition, e.g., a purified or partially purified expressedprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. The methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen as described above. The antigenmay be coupled to carrier molecules such as keyhole limpet hemocyanin ifnecessary. The antigen would typically be mixed with adjuvant, such asFreund's complete or incomplete adjuvant. Booster injections with thesame antigen would occur at approximately two-week intervals. Followingimmunization, somatic cells with the potential for producing antibodies,specifically B lymphocytes (B cells), are selected for use in the MAbgenerating protocol. These cells may be obtained from biopsied spleens,tonsils or lymph nodes, or from a peripheral blood sample. Spleen cellsand peripheral blood cells are preferred, the former because they are arich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easilyaccessible. Often, a panel of animals will have been immunized and thespleen of the animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

The selected hybridomas are then serially diluted and cloned intoindividual antibody-producing cell lines, which clones may then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma may beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines also can be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means can be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Large amounts of the monoclonal antibodies of the present invention alsocan be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals which are histocompatible with the parent cells,e.g., syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonalantibody of the invention may be obtained from the monoclonal antibodyproduced as described above, by methods which include digestion withenzymes such as pepsin or papain and/or cleavage of disulfide bonds bychemical reduction. Alternatively, monoclonal antibody fragmentsencompassed by the present invention may be synthesized using anautomated peptide synthesizer.

Antibodies can also be derived from a recombinant antibody library thatis based on amino acid sequences that have been designed in silico andencoded by polynucleotides that are synthetically generated. Methods fordesigning and obtaining in silico-created sequences are known in the art(Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J.Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).

Digestion of antibodies to produce antigen-binding fragments thereof canbe performed using techniques well known in the art. For example, theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment, which comprises bothantigen-binding sites. “Fv” fragments can be produced by preferentialproteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, butare more commonly derived using recombinant techniques known in the art.The Fv fragment includes a non-covalent V_(H)::V_(L) heterodimerincluding an antigen-binding site which retains much of the antigenrecognition and binding capabilities of the native antibody molecule(Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochmanet al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem.19:4091-4096 (1980)).

Antibody fragments that specifically bind to the polypeptide indicatorsdisclosed herein can also be isolated from a library of scFvs usingknown techniques, such as those described in U.S. Pat. No. 5,885,793.

A wide variety of expression systems are available in the art for theproduction of antibody fragments, including Fab fragments, scFv, VL andVHs. For example, expression systems of both prokaryotic and eukaryoticorigin may be used for the large-scale production of antibody fragments.Particularly advantageous are expression systems that permit thesecretion of large amounts of antibody fragments into the culturemedium. Eukaryotic expression systems for large-scale production ofantibody fragments and antibody fusion proteins have been described thatare based on mammalian cells, insect cells, plants, transgenic animals,and lower eukaryotes. For example, the cost-effective, large-scaleproduction of antibody fragments can be achieved in yeast fermentationsystems. Large-scale fermentation of these organisms is well known inthe art and is currently used for bulk production of several recombinantproteins.

Antibodies that bind to the polypeptide biomarkers employed in thepresent methods are well known to those of skill in the art and in somecases are available commercially or can be obtained without undueexperimentation.

In still other embodiments, particularly where oligonucleotides are usedas binding partners to detect and hybridize to mRNA biomarkers or othernucleic acid based biomarkers, the binding partners (e.g.,oligonucleotides) can comprise a label, e.g., a fluorescent moiety ordye. In addition, any binding partner of the invention, e.g., anantibody, can also be labeled with a fluorescent moiety. A “fluorescentmoiety,” as that term is used herein, includes one or more fluorescententities whose total fluorescence is such that the moiety may bedetected in the single molecule detectors described herein. Thus, afluorescent moiety may comprise a single entity (e.g., a Quantum Dot orfluorescent molecule) or a plurality of entities (e.g., a plurality offluorescent molecules). It will be appreciated that when “moiety,” asthat term is used herein, refers to a group of fluorescent entities,e.g., a plurality of fluorescent dye molecules, each individual entitymay be attached to the binding partner separately or the entities may beattached together, as long as the entities as a group provide sufficientfluorescence to be detected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired limit of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of a molecule, e.g., a marker, at a limitof detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001,0.00001, or 0.000001 pg/ml and with a coefficient of variation of lessthan about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% orless, e.g., about 10% or less, in the instruments described herein. Insome embodiments, the fluorescence of the fluorescent moiety is suchthat it allows detection and/or quantitation of a molecule, e.g., amarker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of lessthan about 10%, in the instruments described herein. “Limit ofdetection,” or LoD, as those terms are used herein, includes the lowestconcentration at which one can identify a sample as containing amolecule of the substance of interest, e.g., the first non-zero value.It can be defined by the variability of zeros and the slope of thestandard curve. For example, the limit of detection of an assay may bedetermined by running a standard curve, determining the standard curvezero value, and adding 2 standard deviations to that value. Aconcentration of the substance of interest that produces a signal equalto this value is the “lower limit of detection” concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties that are preferred arefluorescent moieties, e.g., dye molecules that have a combination of 1)high absorption coefficient; 2) high quantum yield; 3) highphotostability (low photobleaching); and 4) compatibility with labelingthe molecule of interest (e.g., protein) so that it may be analyzedusing the analyzers and systems of the invention (e.g., does not causeprecipitation of the protein of interest, or precipitation of a proteinto which the moiety has been attached).

Any suitable fluorescent moiety may be used. Examples include, but arenot limited to, Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.). TheAlexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874;6,130,101; and 6,974,305 which are herein incorporated by reference intheir entirety. Some embodiments of the invention utilize a dye chosenfrom the group consisting of Alexa Fluor 647, Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, AlexaFluor 700, and Alexa Fluor 750. Some embodiments of the inventionutilize a dye chosen from the group consisting of Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Someembodiments of the invention utilize a dye chosen from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, AlexaFluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Someembodiments of the invention utilize the Alexa Fluor 647 molecule, whichhas an absorption maximum between about 650 and 660 nm and an emissionmaximum between about 660 and 670 nm. The Alexa Fluor 647 dye is usedalone or in combination with other Alexa Fluor dyes.

In some embodiments, the fluorescent label moiety that is used to detectan indicator in a sample using the analyzer systems of the invention isa quantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which aresimilar to the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One optical feature of small excitonic QDs is coloration,which is determined by the size of the dot. The larger the dot, theredder, or more towards the red end of the spectrum the fluorescence.The smaller the dot, the bluer or more towards the blue end it is. Thebandgap energy that determines the energy and hence the color of thefluoresced light is inversely proportional to the square of the size ofthe QD. Larger QDs have more energy levels which are more closelyspaced, thus allowing the QD to absorb photons containing less energy,i.e., those closer to the red end of the spectrum. Because the emissionfrequency of a dot is dependent on the bandgap, it is possible tocontrol the output wavelength of a dot with extreme precision. In someembodiments the protein that is detected with the single moleculeanalyzer system is labeled with a QD. In some embodiments, the singlemolecule analyzer is used to detect a protein labeled with one QD andusing a filter to allow for the detection of different proteins atdifferent wavelengths.

E. Isolated Macromolecular Indicators of Blood Glucose

1. Isolated Polypeptide Indicators

One aspect of the invention pertains to isolated indicator proteins andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against anindicator protein or a fragment thereof. In one embodiment, the nativeindicator protein can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, a protein or peptide comprising thewhole or a segment of the indicator protein is produced by recombinantDNA techniques. Alternative to recombinant expression, such protein orpeptide can be synthesized chemically using standard peptide synthesistechniques. Recombinant proteins can be modified, e.g. glycated, toprovide appropriate antigens for detection of HbA1c. Similarly,non-glycated fragments of hemoglobin can be used to raise antibodiesthat bind either non-glycated hemoglobin alone or total hemoglobin.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of an indicator protein includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the indicator protein, whichinclude fewer amino acids than the full length protein, and exhibit atleast one activity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of an indicator protein can be a polypeptide which is,for example, 10, 25, 50, 100 or more amino acids in length. Moreover,other biologically active portions, in which other regions of the markerprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the nativeform of the indicator protein.

Preferred indicator proteins are encoded by nucleotide sequencesprovided in the sequence listing. Other useful proteins aresubstantially identical (e.g., at least about 40%, preferably 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to one ofthese sequences and retain the functional activity of the correspondingnaturally-occurring indicator protein yet differ in amino acid sequencedue to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position.Preferably, the percent identity between the two sequences is calculatedusing a global alignment. Alternatively, the percent identity betweenthe two sequences is calculated using a local alignment. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. In another embodiment, the two sequences are not the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the BLASTN and BLASTX programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the BLASTPprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, a newer version of the BLASTalgorithm called Gapped BLAST can be utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to performgapped local alignments for the programs BLASTN, BLASTP and BLASTX.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

Another aspect of the invention pertains to antibodies directed againsta protein of the invention. In preferred embodiments, the antibodiesspecifically bind a marker protein or a fragment thereof. The terms“antibody” and “antibodies” as used interchangeably herein refer toimmunoglobulin molecules as well as fragments and derivatives thereofthat comprise an immunologically active portion of an immunoglobulinmolecule, (i.e., such a portion contains an antigen binding site whichspecifically binds an antigen, such as a marker protein, e.g., anepitope of a marker protein). An antibody which specifically binds to aprotein of the invention is an antibody which binds the protein, butdoes not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the protein. Examples of animmunologically active portion of an immunoglobulin molecule include,but are not limited to, single-chain antibodies (scAb), F(ab) andF(ab′)₂ fragments.

An isolated protein of the invention or a fragment thereof can be usedas an immunogen to generate antibodies. The full-length protein can beused or, alternatively, the invention provides antigenic peptidefragments for use as immunogens. The antigenic peptide of a protein ofthe invention comprises at least 8 (preferably 10, 15, 20, or 30 ormore) amino acid residues of the amino acid sequence of one of theproteins of the invention, and encompasses at least one epitope of theprotein such that an antibody raised against the peptide forms aspecific immune complex with the protein. In certain embodiments, theprotein is post-translationally modified. Preferred epitopes encompassedby the antigenic peptide are regions that are located on the surface ofthe protein, e.g., hydrophilic regions. Hydrophobicity sequenceanalysis, hydrophilicity sequence analysis, or similar analyses can beused to identify hydrophilic regions. In preferred embodiments, anisolated marker protein or fragment thereof is used as an immunogen.

The invention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. Preferred polyclonal and monoclonal antibodycompositions are ones that have been selected for antibodies directedagainst a protein of the invention. Particularly preferred polyclonaland monoclonal antibody preparations are ones that contain onlyantibodies directed against a marker protein or fragment thereof.Methods of making polyclonal, monoclonal, and recombinant antibody andantibody fragments are well known in the art.

2. Isolated Nucleic Acid Indicators

One aspect of the invention pertains to isolated nucleic acid molecules,including nucleic acids which encode Eno1 or a portion thereof. Isolatednucleic acids of the invention also include nucleic acid moleculessufficient for use as hybridization probes to identify Eno1 nucleic acidmolecules, and fragments thereof, e.g., those suitable for use as PCRprimers for the amplification of a specific product or mutation ofmarker nucleic acid molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In one embodiment, an “isolated” nucleic acidmolecule (preferably a protein-encoding sequences) is free of sequenceswhich naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. In anotherembodiment, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. A nucleic acid molecule that is substantially free ofcellular material includes preparations having less than about 30%, 20%,10%, or 5% of heterologous nucleic acid (also referred to herein as a“contaminating nucleic acid”).

A nucleic acid molecule of the present invention can be isolated usingstandard molecular biology techniques and the sequence information inthe database records described herein. Using all or a portion of suchnucleic acid sequences, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., ed., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, nucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises an Eno1 molecule which has a nucleotide sequencecomplementary to the nucleotide sequence of a marker nucleic acid or tothe nucleotide sequence of a nucleic acid encoding Eno1. A nucleic acidmolecule which is complementary to a given nucleotide sequence is onewhich is sufficiently complementary to the given nucleotide sequencethat it can hybridize to the given nucleotide sequence thereby forming astable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises an Eno1 nucleic acid or which encodes an Eno1protein. Such nucleic acids can be used, for example, as a probe orprimer. The probe/primer typically is used as one or more substantiallypurified oligonucleotides. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 15, more preferably at least about 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of Eno1.

Probes based on the sequence of Eno1 can be used to detect transcriptsor genomic sequences corresponding to Eno1. In certain embodiments, theprobes hybridize to nucleic acid sequences that traverse splicejunctions. The probe comprises a label group attached thereto, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as part of a diagnostic test kit or panel foridentifying cells, tissues, or individuals which express or mis-expressthe Eno1 protein, such as by measuring levels of a nucleic acid moleculeencoding Eno1 in a sample from a subject, e.g., detecting mRNA levels ordetermining whether a gene encoding Eno1 or its translational controlsequences have been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ,due to degeneracy of the genetic code, from the nucleotide sequence ofnucleic acids encoding Eno1 protein (e.g., protein having the sequenceprovided in the sequence listing), and thus encode the same protein.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence can existwithin a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

As used herein, the phrase “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to an indicator of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, or more nucleotides in lengthand hybridizes under stringent conditions to an Eno1 nucleic acid or toa nucleic acid encoding Eno1. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%(65%, 70%, preferably 75%) identical to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in sections 6.3.1-6.3.6 of CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

F. Indicator Applications

The invention provides methods for diagnosing elevated blood glucose,e.g., pre-diabetes, type 2 diabetes, type 1 diabetes, gestationaldiabetes, in a subject. The invention further provides methods forprognosing or monitoring progression or monitoring response of a subjectwith elevated blood glucose to a therapeutic treatment.

In one aspect, the present invention constitutes an application ofdiagnostic information obtainable by the methods of the invention inconnection with analyzing, detecting, and/or measuring the level of Eno1with at least one other indicator of blood glucose, e.g., blood glucose,e.g., fed blood glucose, fasting blood glucose, glucose tolerance,ketone level; and Hb1Ac levels.

For example, when executing the methods of the invention for detectingand/or measuring a polypeptide indicator, as described herein, onecontacts a biological sample with a detection reagent, e.g, a monoclonalantibody, which selectively binds to the indicator of interest, forminga protein-protein complex, which is then further detected eitherdirectly (if the antibody comprises a label) or indirectly (if asecondary detection reagent is used, e.g., a secondary antibody, whichin turn is labeled). Thus, the method of the invention transforms thepolypeptide indicators of the invention to a protein-protein complexthat comprises either a detectable primary antibody or a primary andfurther secondary antibody. Forming such protein-protein complexes isrequired in order to identify the presence of the biomarker of interestand necessarily changes the physical characteristics and properties ofthe indicator of interest as a result of conducting the methods of theinvention.

The same principal applies when conducting the methods of the inventionfor detecting Eno1 nucleic acids. In particular, when amplificationmethods are used to detect an Eno1 mRNA, the amplification process, infact, results in the formation of a new population of amplicons—i.e.,molecules that are newly synthesized and which were not present in theoriginal biological sample, thereby physically transforming thebiological sample. Similarly, when hybridization probes are used todetect Eno1, a physical new species of molecules is in effect created bythe hybridization of the probes (optionally comprising a label) to thetarget biomarker mRNA (or other nucleic acid), which is then detected.Such polynucleotide products are effectively newly created or formed asa consequence of carrying out the method of the invention.

The invention provides, in one embodiment, methods for diagnosingelevated blood glucose, e.g., pre-diabetes, diabetes, e.g., type 2diabetes, type 1 diabetes, gestational diabetes. The methods of thepresent invention can be practiced in conjunction with any other methodused by the skilled practitioner to prognose the occurrence orrecurrence of elevated blood glucose and/or the response to atherapeutic intervention of a subject being treated for elevated bloodglucose. The diagnostic and prognostic methods provided herein can beused to determine if additional and/or more complex or cumbersome testsor monitoring (e.g., glucose tolerance test, continuous glucosemonitoring) should be performed on a subject. It is understood that adisease as complex as pre-diabetes or diabetes is rarely diagnosed usinga single test. Therefore, it is understood that the diagnostic,prognostic, and monitoring methods provided herein are typically used inconjunction with other methods known in the art. For example, themethods for detection of the level of Eno1 as provided by the inventionmay be performed in conjunction with a detection of Hb1Ac levels,detection of blood glucose levels under fasting or fed conditions, orglucose tolerance test.

Methods for assessing the efficacy of a treatment regimen, e.g., drugtreatment, behavior modification, surgery, or any other therapeuticapproach useful for treating elevated blood glucose in a subject arealso provided. In these methods the amount of Eno1 in a pair of samples(a first sample obtained from the subject at an earlier time point orprior to the treatment regimen and a second sample obtained from thesubject at a later time point, e.g., at a later time point when thesubject has undergone at least a portion of the treatment regimen) isassessed. It is understood that the methods of the invention includeobtaining and analyzing more than two samples (e.g., 3, 4, 5, 6, 7, 8,9, or more samples) at regular or irregular intervals for assessment ofmarker levels. Pairwise comparisons can be made between consecutive ornon-consecutive subject samples. Trends of marker levels and rates ofchange of marker levels can be analyzed for any two or more consecutiveor non-consecutive subject samples. Measurement of Eno1 levels can beperformed in conjunction with other methods for the detection anmonitoring of blood glucose.

The methods of the invention may also be used to select a compound thatis capable of modulating blood glucose by modulation of Eno1 expressionor activity. In this method, a cell, preferably a cell with alteredinsulin sensitivity or altered glucose uptake is contacted with a testcompound, and the ability of the test compound to modulate theexpression and/or activity of Eno1 in the cell is determined, therebyselecting a compound that is capable of modulating Eno1 expression oractivity, preferably increasing Eno1 expression or activity therebyincreasing glucose uptake in the cell.

Using the methods described herein, a variety of molecules, may bescreened in order to identify molecules which modulate, preferablyincrease the expression and/or activity of Eno1. Compounds so identifiedcan be provided to a subject in order to normalize blood glucose by oneor more of increasing glucose uptake, increasing insulin sensitivity,and/or decreasing insulin resistance thereby treating elevated bloodglucose, e.g., pre-diabetes or diabetes, e.g., type 2 diabetes, type 1diabetes, or gestational diabetes.

The present invention pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningthe level of expression of Eno1 protein or nucleic acid, in order todetermine whether an individual is at risk of developing a disease ordisorder related to elevated blood glucose, such as, without limitation,pre-diabetes or diabetes including type 2 diabetes, type 1 diabetes, orgestational diabetes. Such assays can be used for prognostic orpredictive purposes to thereby prophylactically treat an individualprior to the onset of the disorder.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other therapeutic compounds) or behavioraland/or diet modifications on the expression or activity of Eno1 inclinical trials. These and other applications are described in furtherdetail in the following sections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence or change ofan indicator protein or nucleic acid in a biological sample involvesobtaining a biological sample (e.g. blood or serum) from a test subjectand contacting the biological sample with a compound or an agent capableof detecting the polypeptide or nucleic acid (e.g., mRNA or cDNA). Thedetection methods of the invention can thus be used to detect mRNA,cDNA, or protein including post-translationally modified proteins, forexample, in a biological sample in vitro as well as in vivo.

Methods provided herein for detecting the presence, absence, change ofthe level of an indicator protein or nucleic acid in a biological sampleinclude obtaining a biological sample from a subject that may or may notcontain the marker protein or nucleic acid to be detected, contactingthe sample with an indicator-specific binding agent (i.e., one or moremarker-specific binding agents) that is capable of forming a complexwith the indicator protein or nucleic acid to be detected, andcontacting the sample with a detection reagent for detection of theindicator—indicator-specific binding agent complex, if formed. It isunderstood that the methods provided herein for detecting a level of anindicator in a biological sample includes the steps to perform theassay. In certain embodiments of the detection methods, the level of theindicator protein or nucleic acid in the sample is none or below thethreshold for detection.

The methods include formation of either a transient or stable complexbetween the indicator and the indicator-specific binding agent. Themethods require that the complex, if formed, be formed for sufficienttime to allow a detection reagent to bind the complex and produce adetectable signal (e.g., fluorescent signal, a signal from a product ofan enzymatic reaction, e.g., a peroxidase reaction, a phosphatasereaction, a beta-galactosidase reaction, or a polymerase reaction).

In certain embodiments, all of the indicators are detected using thesame method. In certain embodiments, all of the indicators are detectedusing the same biological sample (e.g., same body fluid). In certainembodiments, different indicators are detected using different methods.In certain embodiments, indicators are detected in different biologicalsamples (e.g., blood and serum).

2. Protein Detection

In certain embodiments of the invention, the indicator to be detected isa protein. In certain embodiments, the indicator to be detected is apost-translationally modified protein. Proteins are detected using anumber of assays in which a complex between the indicator protein to bedetected and the indicator specific binding agent would not occurnaturally, for example, because one of the components is not a naturallyoccurring compound or the indicator for detection and the indicatorspecific binding agent are not from the same organism (e.g., humanindicator proteins detected using indicator-specific binding antibodiesfrom mouse, rat, or goat). In a preferred embodiment of the invention,the indicator protein for detection is a human indicator protein. Incertain detection assays, the human indicators for detection are boundby indicator-specific, non-human antibodies, thus, the complex would notbe formed in nature. The complex of the indicator protein can bedetected directly, e.g., by use of a labeled indicator-specific antibodythat binds directly to the indicator, or by binding a further componentto the indicator—indicator-specific antibody complex. In certainembodiments, the further component is a second indicator-specificantibody capable of binding the indicator at the same time as the firstindicator-specific antibody. In certain embodiments, the furthercomponent is a secondary antibody that binds to an indicator-specificantibody, wherein the secondary antibody preferably linked to adetectable label (e.g., fluorescent label, enzymatic label, biotin).When the secondary antibody is linked to an enzymatic detectable label(e.g., a peroxidase, a phosphatase, a beta-galactosidase), the secondaryantibody is detected by contacting the enzymatic detectable label withan appropriate substrate to produce a colorimetric, fluorescent, orother detectable, preferably quantitatively detectable, product.Antibodies for use in the methods of the invention can be polyclonal,however, in a preferred embodiment monoclonal antibodies are used. Anintact antibody, or a fragment or derivative thereof (e.g., Fab orF(ab′)₂) can be used in the methods of the invention. Such strategies ofindicator protein detection are used, for example, in ELISA, RIA,western blot, and immunofluorescence assay methods.

In certain detection assays, the indicator present in the biologicalsample for detection is an enzyme, e.g., Eno1, and the detection reagentis an enzyme substrate (e.g., 2-phosphoglycerate (2-PG) orphosphoenolpyruvate (PEP), or an analog of either of the compounds thatproduces a detectable product). In preferred embodiments, the substratewhich forms a complex with the indicator enzyme to be detected is notthe substrate for the enzyme in a human subject.

In certain embodiments, the indicator—indicator-specific binding agentcomplex is attached to a solid support for detection of the indicator.The complex can be formed on the substrate or formed prior to capture onthe substrate. For example, in an ELISA, RIA, immunoprecipitation assay,western blot, immunofluorescence assay, in gel enzymatic assay theindicator for detection is attached to a solid support, either directlyor indirectly. In an ELISA, RIA, or immunofluorescence assay, theindicator is typically attached indirectly to a solid support through anantibody or binding protein. In a western blot or immunofluorescenceassay, the indicator is typically attached directly to the solidsupport. For in-gel enzyme assays, the indicator is resolved in a gel,typically an acrylamide gel, in which a substrate for the enzyme isintegrated.

3. Nucleic Acid Detection

In certain embodiments of the invention, the indicator is a nucleicacid, e.g., an Eno1 nucleic acid. Nucleic acids are detected using anumber of assays in which a complex between the indicator nucleic acidto be detected and an indicator-specific probe would not occurnaturally, for example, because one of the components is not a naturallyoccurring compound. In certain embodiments, the analyte comprises anucleic acid and the probe comprises one or more synthetic singlestranded nucleic acid molecules, e.g., a DNA molecule, a DNA-RNA hybrid,a PNA, or a modified nucleic acid molecule containing one or moreartificial bases, sugars, or backbone moieties. In certain embodiments,the synthetic nucleic acid is a single stranded is a DNA molecule thatincludes a fluorescent label. In certain embodiments, the syntheticnucleic acid is a single stranded oligonucleotide molecule of about 12to about 50 nucleotides in length. In certain embodiments, the nucleicacid to be detected is an mRNA and the complex formed is an mRNAhybridized to a single stranded DNA molecule that is complementary tothe mRNA. In certain embodiments, an RNA is detected by generation of aDNA molecule (i.e., a cDNA molecule) first from the RNA template usingthe single stranded DNA that hybridizes to the RNA as a primer, e.g., ageneral poly-T primer to transcribe poly-A RNA. The cDNA can then beused as a template for an amplification reaction, e.g., PCR, primerextension assay, using a marker-specific probe. In certain embodiments,a labeled single stranded DNA can be hybridized to the RNA present inthe sample for detection of the RNA by fluorescence in situhybridization (FISH) or for detection of the RNA by northern blot.

For example, in vitro techniques for detection of mRNA include northernhybridizations, in situ hybridizations, and rtPCR. In vitro techniquesfor detection of genomic DNA include Southern hybridizations. Techniquesfor detection of mRNA include PCR, northern hybridizations, and in situhybridizations. Methods include both qualitative and quantitativemethods.

A general principle of such diagnostic, prognostic, and monitoringassays involves preparing a sample or reaction mixture that may containa nucleic acid for detection, and a probe, under appropriate conditionsand for a time sufficient to allow the indicator nucleic acid and probeto interact and bind, thus forming a complex that can be removed and/ordetected in the reaction mixture. These assays can be conducted in avariety of ways known in the art, e.g., PCR, FISH, northern blot.

4. Detection of Expression Levels

Eno1 levels can be detected based on the absolute expression level or anormalized or relative expression level. Detection of absolute Eno1levels may be preferable when monitoring the treatment of a subject orin determining if there is a change in the blood glucose level or bloodglucose regulation in a subject. For example, the expression level ofEno1 can be monitored in a subject undergoing treatment for abnormalblood glucose, e.g., at regular intervals, such a monthly intervals. Amodulation in the level of Eno1 can be monitored over time to observetrends in changes in Eno1 levels. The expression level of Eno1 in thesubject may be higher than the expression level of Eno1 in a normalsample, but may be higher than the prior expression level, thusindicating a benefit of the treatment regimen for the subject.Similarly, rates of change of an Eno1 level can be important in asubject who is being treated with behavior or diet modification ratherthan therapeutic interventions. Changes, or no changes, in Eno1 levelsin an individual subject may be more relevant to treatment decisions forthe subject than Eno1 levels present in the population. Rapid changes inEno1 levels in a subject who otherwise appears to have a normal bloodglucose may be indicative of an abnormal blood glucose or apredisposition to develop a condition related to abnormal blood glucose,even if the markers are within normal ranges for the population. Eno1level can be determined or monitored in conjunction with one or moreadditional indicators of elevated blood glucose, e.g., HbA1c, increasedblood glucose including one or more of increased fed or fasting bloodglucose, or decreased rate of glucose clearance in a glucose tolerancetest.

As an alternative to making determinations based on the absoluteexpression level of Eno1, determinations may be based on the normalizedexpression level of Eno1. Expression levels are normalized by comparingthe absolute expression level of an indicator to the expression of agene that is not an indicator, e.g., a housekeeping gene that isconstitutively expressed. Suitable genes for normalization includehousekeeping genes such as the actin gene and suitable proteins fornormalization in blood or serum include albumin. This normalizationallows the comparison of the expression level in one sample, e.g., asample from a subject with normal blood glucose, to another sample,e.g., a sample from a subject suspected of having or having abnormalblood glucose, or between samples from different sources.

Alternatively, the expression level can be provided as a relativeexpression level as compared to an appropriate control, e.g., populationcontrol, earlier time point control, etc. Preferably, the samples usedin the baseline determination will be from samples from subjects withnormal blood glucose. The choice of the cell source is dependent on theuse of the relative expression level. In addition, as more data isaccumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.

5. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof an indicator of blood glucose can be applied not only in basic drugscreening or monitoring the treatment of a single subject, but also inclinical trials. For example, the effectiveness of an agent to affectEno1 expression can be monitored in clinical trials of subjectsreceiving treatment for elevated blood glucose. In a preferredembodiment, the present invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of the indicator Eno1and optionally one or more further indicators of blood glucose, e.g.,blood glucose, ketone, or HbA1c in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression of the indicator(s) in thepost-administration samples; (v) comparing the level of indicator(s) inthe pre-administration sample with the level of the indicator(s) in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,decreased Eno1 expression and lack of normalization of otherindicator(s) during the course of treatment may indicate ineffectivedosage and the desirability of increasing the dosage. Conversely,increased expression of Eno1 and normalization of other indicator(s) mayindicate efficacious treatment and no need to change dosage.

VI. TREATMENT OF IMPAIRED BLOOD GLUCOSE LEVELS, IMPAIRED BLOOD GLUCOSELEVEL CONTROL, AND DIABETES

As demonstrated herein, administration of Eno1 protein improves glucoseuptake and response, normalizing blood glucose levels and control ofblood glucose levels. The invention provides methods of treatment ofsubjects suffering from impaired glucose tolerance, increased bloodglucose, insulin resistance, insulin insufficiency, and diabetes, e.g.,type 2 diabetes, type 1 diabetes, pre-diabetes, and gestational diabetesby administering Eno1 to the subject to ameliorate at least one sign orsymptom of the conditions. In certain embodiments, Eno1, preferablytranscript variant 1 of Eno1, can be administered to a subject whereinat least one additional agent for the treatment of impaired glucosetolerance, increased blood glucose, insulin resistance, insulininsufficiency, or diabetes is administered to the subject. As usedherein, the agents can be administered sequentially, in either order, orat the same time. Administration of multiple agents to a subject doesnot require co-formulation of the agents or the same administrationregimen.

The method of treatment of impaired glucose tolerance, increased bloodglucose, insulin resistance, insulin insufficiency, or diabetes,especially type 2 diabetes, using Eno1 can be combined with knownmethods and agents for the treatment of diabetes. Many agents andregimens are currently available for treatment of diabetes. The specificagent selected for treatment depends upon the subject, the specificsymptoms and the severity of the disease state. For example, in certainembodiments, Eno1 can be administered in conjunction with dietary and/orbehavior modification, e.g., caloric restriction, alone or incombination with bariatric surgery, and/or with increased physicalactivity. In certain embodiments, Eno1 can be administered with agentsfor the treatment of type 2 diabetes, e.g., metformin (Glucophage,Glumetza, others), glitazones, e.g., pioglitazone (Actos), glipizide(Glucotrol), glyburide (Diabeta, Glynase), glimepiride (Amaryl),acarbose (Precose), metformin (Glucophage), Sitagliptin (Januvia),Saxagliptin (Onglyza), Repaglinide (Prandin), Nateglinide (Starlix),Exenatide (Byetta), Liraglutide (Victoza), or insulin. Insulins aretypically used only in treatment of later stage type 2 diabetes andinclude rapid-acting insulin (insulin aspart (NovoLog), insulinglulisine (Apidra), and insulin lispro (Humalog)); short-acting insulin(insulin regular (Humulin R, Novolin R)); intermediate-acting insulin(insulin NPH human (Humulin N, Novolin N)), and long-acting insulin(insulin glargine (Lantus) and insulin detemir (Levemir)). Treatmentsfor diabetes can also include behavior modification including exerciseand weight loss which can be facilitated by the use of drugs or surgery.Treatments for elevated blood glucose and diabetes can be combined. Forexample, drug therapy can be combined with behavior modificationtherapy. Insulins for use in treatment of type 1 diabetes include, butare not limited to Insulins are typically used only in treatment oflater stage type 2 diabetes and include rapid-acting insulin (insulinaspart (NovoLog), insulin glulisine (Apidra), and insulin lispro(Humalog)); short-acting insulin (insulin regular (Humulin R, NovolinR)); intermediate-acting insulin (insulin NPH human (Humulin N, NovolinN)), and long-acting insulin (insulin glargine (Lantus) and insulindetemir (Levemir)).

Accordingly, in some aspects, the invention relates to a method oftreating elevated blood glucose in a subject, comprising: (a) obtaininga biological sample from a subject suspected of having elevated bloodglucose, (b) submitting the biological sample to obtain diagnosticinformation as to the level of Eno1, and (c) administering atherapeutically effective amount of an anti-diabetic therapy to thesubject when the level of Eno1 in the sample is above a threshold level.

In some aspects, the invention relates to a method of treating elevatedblood glucose in a subject, comprising: (a) obtaining diagnosticinformation as to the level of Eno1 in a biological sample from thesubject, and (b) administering a therapeutically effective amount of ananti-diabetic therapy to the subject when the level of Eno1 in thesample is above a threshold level.

In some aspects, the invention relates to a method of treating elevatedblood glucose in a subject, comprising: (a) obtaining a biologicalsample from a subject suspected of having elevated blood glucose for usein identifying diagnostic information as to the level of Eno1, (b)detecting the level of Eno1 in the biological sample, (c) recommendingto a healthcare provider to administer a blood glucose lowering therapyto the subject when the level of Eno1 in the sample is below a thresholdlevel.

The methods described above may further comprising obtaining diagnosticinformation as to the level of one or more additional indicators ofelevated blood glucose. In some embodiments the methods further comprisemeasuring a level of one or more additional indicators of elevated bloodglucose. The one or more additional indicators of elevated blood glucosemay be selected from the group consisting of HbA1c level, fastingglucose level, fed glucose level, and glucose tolerance.

In some embodiments of the aforementioned methods, step (c) furthercomprises administering a therapeutically effective amount of a glucoselowering therapy to the subject if the level of Eno1 in the sample isbelow a threshold level and at least one of the additional indicators ofelevated blood glucose is detected. In some embodiments step (c) furthercomprises recommending to a healthcare provider to administer a glucoselowering therapy to the subject if the level of Eno1 in the sample isbelow a threshold level and at least one of the additional indicators ofelevated blood glucose is detected.

In some embodiments of the methods described above, the biologicalsample is blood or serum. In some embodiments, the level of Eno1 isdetermined by immunoassay or ELISA. In some embodiments, the level ofEno1 is determined by (i) contacting the biological sample with areagent that selectively binds to the Eno1 to form a biomarker complex,and (ii) detecting the biomarker complex. In some embodiments, thereagent that selectively binds to the Eno1 to form a biomarker complexis an anti-Eno1 antibody that selectively binds to at least one epitopeof Eno1.

In some embodiments of the methods described above, the level of Eno1 isdetected by measuring the amount of Eno1 mRNA in the biological sample.The amount of Eno1 mRNA may be detected, for example, by anamplification reaction. In some embodiments, the amplification reactionis (a) a polymerase chain reaction (PCR); (b) a nucleic acidsequence-based amplification assay (NASBA); (c) a transcription mediatedamplification (TMA); (d) a ligase chain reaction (LCR); or (e) a stranddisplacement amplification (SDA).

In some embodiments, a hybridization assay is used for detecting theamount of Eno1 mRNA in the biological sample. In some embodiments, anoligonucleotide that is complementary to a portion of a Eno1 mRNA isused in the hybridization assay to detect the Eno1 mRNA.

VI. ANIMAL MODELS OF DIABETES AND INSULIN RESISTANCE

A number of genetic and induced animal models of metabolic syndromessuch as type 1 and type 2 diabetes, insulin resistance, hyperlipidemia,are well characterized in the art. Such animals can be used todemonstrate the effect of Eno1 in the treatment of insulin resistanceand diabetes. Models of type 1 diabetes include, but are not limited to,NOD mice and streptozotocin-induced diabetes in rats and mice (models oftype 1 diabetes). Genetic and induced models of type 2 diabetes include,but are not limited to, the leptin deficient ob/ob mouse, the leptinreceptor deficient db/db mouse, and high fat fed mouse or rat models. Ineach of the models, the timeline for development of specific diseasecharacteristics are well known. Eno1 can be administered before or afterthe appearance of symptoms of diabetes or insulin resistance todemonstrate the efficacy of Eno1 in the prevention or treatment ofdiabetes and/or insulin resistance in these animal models.

Depending on the specific animal model selected and the time ofintervention, e.g., before or after the appearance of diabetes and/orinsulin resistance, the animal models can be used to demonstrate theefficacy of the methods provide herein for the prevention, treatment,diagnosis, and monitoring of diabetes and/or insulin resistance.

VII. DRUG SCREENING

Administration of Eno1 results in normalization of blood glucose inanimals with induced diabetes, making Eno1 an attractive targets foridentification of new therapeutic agents via screens to detect compoundsor entities that enhance expression of Eno1. Accordingly, the presentinvention provides methods for the identification of compoundspotentially useful for modulating blood glucose and diabetes. Inparticular, the present invention provides methods for theidentification of compounds potentially useful for modulating Eno1wherein the compounds modulate blood glucose and diabetes.

Such assays typically comprise a reaction between Eno1 and one or moreassay components, e.g., test compounds. The other components may beeither a test compound itself, or a combination of test compounds and anatural binding partner of Eno1. Compounds identified via assays such asthose described herein may be useful, for example, for modulating, e.g.,inhibiting, ameliorating, treating, or preventing the disease. Compoundsidentified for modulating the expression level of Eno1 are preferablyfurther tested for activity useful in the treatment of abnormal bloodglucose and/or diabetes, e.g., normalizing fed and/or fasting glucose,normalizing glucose clearance and/or insulin levels in a glucosetolerance test, normalizing HbA1c levels.

The test compounds used in the screening assays of the present inventionmay be obtained from any available source, including systematiclibraries of natural and/or synthetic compounds. Test compounds may alsobe obtained by any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library and peptoid libraryapproaches are limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

The screening methods of the invention comprise contacting a cell, e.g.,a diseased cell, especially a cell with abnormal insulin response and/orglucose uptake, with a test compound and determining the ability of thetest compound to modulate the expression and/or activity of Eno1 in thecell. The expression and/or activity of Eno1, optionally in combinationwith methods of detection of blood glucose levels, can be determinedusing any methods known in the art, such as those described herein.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of Eno1 or biologicallyactive portions thereof. In yet another embodiment, the inventionprovides assays for screening candidate or test compounds which bind toEno1 or biologically active portions thereof. Determining the ability ofthe test compound to directly bind to Eno1 can be accomplished, forexample, by any method known in the art.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent capable of modulatingthe expression and/or activity of Eno1 can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatment asdescribed above.

In certain embodiments, the screening methods are performed using cellscontained in a plurality of wells of a multi-well assay plate. Suchassay plates are commercially available, for example, from StratageneCorp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.) and include,for example, 48-well, 96-well, 384-well and 1536-well plates.

Reproducibility of the results may be tested by performing the analysismore than once with the same concentration of the same candidatecompound (for example, by incubating cells in more than one well of anassay plate). Additionally, since candidate compounds may be effectiveat varying concentrations depending on the nature of the compound andthe nature of its mechanism(s) of action, varying concentrations of thecandidate compound may be tested. Generally, candidate compoundconcentrations from 1 fM to about 10 mM are used for screening.Preferred screening concentrations are generally between about 10 pM andabout 100 μm.

The screening methods of the invention will provide “hits” or “leads,”i.e., compounds that possess a desired but not optimized biologicalactivity. Lead optimization performed on these compounds to fulfill allphysicochemical, pharmacokinetic, and toxicologic factors required forclinical usefulness may provide improved drug candidates. The presentinvention also encompasses these improved drug candidates and their useas therapeutics for modulating blood glucose and insulin response.

VIII. KITS/PANELS

The invention also provides compositions and kits for diagnosing,prognosing, or monitoring a disease or disorder, recurrence of adisorder, or survival of a subject being treated for a disorder (e.g.,abnormal blood glucose and/or diabetes). These kits include one or moreof the following: a detectable antibody that specifically binds to Eno1,a detectable antibody that specifically binds to Eno1, reagents forobtaining and/or preparing subject tissue samples for staining, andinstructions for use.

The invention also encompasses kits for detecting the presence of Eno1protein or nucleic acid in a biological sample. Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping an abnormal blood glucose and/or diabetes. For example, thekit can comprise a labeled compound or agent capable of detecting Eno1protein or nucleic acid in a biological sample and means for determiningthe amount of the protein or mRNA in the sample (e.g., an antibody whichbinds the protein or a fragment thereof, or an oligonucleotide probewhich binds to DNA or mRNA encoding the protein). Kits can also includeinstructions for use of the kit for practicing any of the methodsprovided herein or interpreting the results obtained using the kit basedon the teachings provided herein. The kits can also include reagents fordetection of a control protein in the sample not related to abnormalblood glucose, e.g., actin for tissue samples, albumin in blood or bloodderived samples for normalization of the amount of the Eno1 present inthe sample. The kit can also include the purified marker for detectionfor use as a control or for quantitation of the assay performed with thekit.

Kits include a panel of reagents for use in a method to diagnoseabnormal blood glucose in a subject (or to identify a subjectpredisposed to developing abnormal blood glucose and/or diabetes), thepanel comprising at least two detection reagents comprising a reagentfor detection of Eno1 level and a reagent for detection of anotherindicator of blood glucose, e.g., HbA1c.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to a Eno1; and,optionally, (2) a second, different antibody which binds to either Eno1or the first antibody and is conjugated to a detectable label. Incertain embodiments, the kit includes (1) a second antibody (e.g.,attached to a solid support) which binds to a second marker protein;and, optionally, (2) a second, different antibody which binds to eitherHbA1c or hemoglobin (either total or unmodified hemoglobin) or thesecond antibody and is conjugated to a detectable label. The first andsecond marker proteins are different.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding Eno1 or (2) a pair ofprimers useful for amplifying an Eno1 nucleic acid molecule. In certainembodiments, the kit includes a third primer specific for each nucleicacid marker to allow for detection using quantitative PCR methods. Incertain embodiments, the kit further includes instructions to measureblood glucose in the subject, either directly or indirectly (e.g., usingHbA1c levels).

For chromatography methods, the kit can include markers, includinglabeled markers, to permit detection and identification of one or moreindicators of blood glucose, e.g., Eno1 and HbA1c by chromatography. Incertain embodiments, kits for chromatography methods include compoundsfor derivatization of one or more blood glucose indicators. In certainembodiments, kits for chromatography methods include columns forresolving the indicators of the method.

Reagents specific for detection of Eno1 allow for detection andquantitation of the marker in a complex mixture, e.g., serum, blood. Incertain embodiments, the reagents are species specific. In certainembodiments, the Eno1 reagents are not species specific. In certainembodiments, the Eno1 reagents are isoform specific. In certainembodiments, the Eno1 reagents are not isoform specific. In certainembodiments, the reagents detect total Eno1.

In certain embodiments, the kits for the diagnosis, monitoring, orcharacterization of elevated blood glucose and/or diabetes comprise atleast one reagent specific for the detection of the level of expressionof Eno1. In certain embodiments, the kits further comprise instructionsto detect the level of blood glucose in a sample, either directly orindirectly, or both. In certain embodiments, the kit includes at leastone reagent for detection of the level of HbA1c.

In certain embodiments, the kits can also comprise, e.g., a bufferingagents, a preservative, a protein stabilizing agent, reaction buffers.The kit can further comprise components necessary for detecting thedetectable label (e.g., an enzyme or a substrate). The kit can alsocontain a control sample or a series of control samples which can beassayed and compared to the test sample. The controls can be controlserum samples or control samples of purified proteins or nucleic acids,as appropriate, with known levels of indicators. Each component of thekit can be enclosed within an individual container and all of thevarious containers can be within a single package, along withinstructions for interpreting the results of the assays performed usingthe kit.

The kits of the invention may optionally comprise additional componentsuseful for performing the methods of the invention.

For example, in some aspects the present invention relates to a kit fordetecting Eno1 in a biological sample comprising at least one reagentfor measuring the level of Eno1 in the biological sample, and a set ofinstructions for measuring the level of Eno1. In some embodiments, thereagent is an anti-Eno1 antibody. In some embodiments, the kit furthercomprises a means to detect the anti-Eno1 antibody. In some embodiments,the means to detect the anti-Eno1 antibody is a detectable secondaryantibody. In some embodiments, the reagent for measuring the level ofEno1 is an oligonucleotide that is complementary to an Eno1 mRNA.

In some embodiments of the aforementioned kits, the instructions setforth an immunoassay or ELISA for detecting the Eno1 level in thebiological sample. In some embodiments, the instructions set forth anamplification reaction for assaying the level of Eno1 mRNA in thebiological sample. In some embodiments, the amplification reaction isused for detecting the amount of Eno1 mRNA in the biological sample. Insome embodiments, the amplification reaction is (a) a polymerase chainreaction (PCR); (b) a nucleic acid sequence-based amplification assay(NASBA); (c) a transcription mediated amplification (TMA); (d) a ligasechain reaction (LCR); or (e) a strand displacement amplification (SDA).

In some embodiments of the aforementioned kits, the instructions setforth a hybridization assay for detecting the amount of Eno1 mRNA in thebiological sample. In some embodiments, the kit further comprises atleast one oligonucleotide that is complementary to a portion of an Eno1mRNA.

The invention further provides panels of reagents for detection of oneor more blood glucose indicators in a subject sample and at least onecontrol reagent. In certain embodiments, the control reagent is todetect the indicator in the biological sample wherein the panel isprovided with a control sample containing the indicator for use as apositive control and optionally to quantitate the amount of indicatorpresent in the biological sample. In certain embodiments, the panelincludes a detection reagent for a protein or nucleic acid not relatedto an abnormal blood glucose that is known to be present or absent inthe biological sample to provide a positive or negative control,respectively. The panel can be provided with reagents for detection of acontrol protein in the sample not related to the abnormal blood glucose,e.g., albumin in blood or blood derived samples for normalization of theamount of the indicator present in the sample. The panel can be providedwith a purified indicator, e.g., Eno1, for detection for use as acontrol or for quantitation of the assay performed with the panel.

In a preferred embodiment, the panel includes reagents for detection ofEno1, preferably in conjunction with a control reagent. In the panel,Eno1 is detected by a reagent specific for that Eno1. In certainembodiments, the panel further includes a reagent for the detection ofHbA1c. In certain embodiments, the panel includes replicate wells,spots, or portions to allow for analysis of various dilutions (e.g.,serial dilutions) of biological samples and control samples. In apreferred embodiment, the panel allows for quantitative detection of oneor more indicators of blood glucose.

In certain embodiments, the panel is a protein chip for detection of oneor more markers. In certain embodiments, the panel is an ELISA plate fordetection of one or more markers. In certain embodiments, the panel is aplate for quantitative PCR for detection of one or more markers.

In certain embodiments, the panel of detection reagents is provided on asingle device including a detection reagent for one or more markers ofthe invention and at least one control sample. In certain embodiments,the panel of detection reagents is provided on a single device includinga detection reagent for two or more markers of the invention and atleast one control sample. In certain embodiments, multiple panels forthe detection of different markers of the invention are provided with atleast one uniform control sample to facilitate comparison of resultsbetween panels.

In certain embodiments, panels and kits further include instructions oradvice for measuring blood glucose in a subject. In certain embodiments,the kit or panel is provided with one or more reagents or devices forthe measurement of blood glucose.

The invention also provides kits for treatment of at least one ofdiabetes, e.g., type 1 diabetes, type 2 diabetes, gestational diabetes,pre-diabetes, insulin resistance, glucose intolerance, abnormal bloodglucose, and loss of blood glucose control. The kits include Eno1 andone or more of instructions for use and a device for administration, asappropriate.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

EXAMPLES Example 1 Employing Platform Technology to Identify Enolase 1(Eno1) as an Important Node of Activity in the Etiology of Diabetes

In this example, the platform technology described in detail ininternational Patent Application No. PCT/US2012/027615 was employed tointegrate data obtained from a custom built diabetes model, and toidentity novel proteins/pathways driving the pathogenesis of diabetes,particularly type 2 diabetes. Relational maps resulting from thisanalysis have identified Eno1 as an important node of activity in theetiology of diabetes. Therefore, Eno1 is an important diabetes treatmenttarget, as well as a diagnostic/prognostic marker associated withdiabetes.

Example 2 Eno1 Regulation of Glucose Uptake in Myotubes

Eno1 was recombinantly expressed in E. coli as a 6×HIS protein tag usinga commercially available expression vector. The tagged Eno1 was purifiedusing affinity chromatography methods known in the art. Preferably, the6×HIS tag was cleaved to produce the protein for use in the methodsprovided herein.

Human skeletal muscle myoblasts (HSMM) were procured from PromoCell andwere cultured in growth media recommended by the vendor. HSMM myoblasts(20,000 cells/well) were differentiated with 2% horse serum in 96 wellplates for 7 days before experiment. Cells were treated with human Eno1(500 ug/ml). Cells were washed twice with 200 μl MBSS modified balancedsalt solution (MBSS) buffer containing 0.1% BSA, and then serum starvedwith 100 ul MBSS 0.1% BSA for 4 hours. Upon initiation of insulinstimulation, 100 ul 2× reagents in MBSS 0.1% BSA buffer was added to 100ul starvation media to make 1× concentration for the experiment. The 2×reagents are: insulin (0, 20 nM, and 200 nM); a fluorescent glucoseanalog 2-NBDG (500 uM). Cells were treated with insulin and thefluorescent deoxy-glucose analog 2-NBDG for 30 min, then washed twicewith MBSS buffer, then 50 ul MBSS buffer were added to wells. Glucoseuptake was detected with fluorometer along with background detectionwith wells with no cells in them. After fluorometer readout, a fixative(formalin, 50 ul) was added to 50 ul MBSS in the wells, then 100 ul 1 uMDAPI was added to 100 ul formalin and MBSS mixture.

As shown in FIGS. 1A and 1B, treatment of myotubes with Eno1significantly increased glucose uptake in both the absence and presenceof insulin (p=0.025 insulin independent glucose uptake untreated vs.Eno1 treated). These results demonstrate a role for Eno1 in both insulindependent and insulin independent glucose uptake. The insulin dependentglucose uptake induced by Eno1 demonstrates that Eno1 is intricatelyconnected with the insulin signaling pathway in at least skeletal musclein subjects sensitive to insulin. The results also demonstrate a rolefor Eno1 in insulin independent glucose uptake. This observation isimportant for treatment of subjects with both type 1 and type 2 diabeteswho suffer from insulin resistance and who may also have hyperinsulinemia, so that insulin action is compromised and hence a insulinindependent. These results demonstrate that Eno1 is useful instimulating glucose uptake even in individuals who no longer have normalinsulin signaling.

Cell cultures of human skeletal muscle myotubes were treated for 48hours with the purified Eno1 protein described above to measure Eno1uptake. Eno1 levels in the cells were then determined by Western blot.As shown in FIGS. 2A and 2B, Eno1 levels in cells treated with 500 μg/mlEno1 or 1000 μg/ml Eno1 had significantly higher levels of Eno1 relativeto untreated cells. Eno1 levels in cells treated with 1000 μg/ml Eno1were also higher than in cells treated with 500 μg/ml Eno1. Theseresults indicate that Eno1 is delivered into human skeletal musclemyotubes in a dose dependent manner.

To determine the role of Eno1 enzyme activity in glucose uptake,purified Eno1 was heat inactivated by treatment at 88° C. for 90seconds, and activity levels of native and heat inactivated Eno1 werecompared. Eno1 activity was determined by colorimetric assay using theEno1 human activity assay kit from Abcam (Cambridge, Mass.; Cat. No.ab117994). As shown in FIG. 3A, heat inactivation greatly reduced Eno1enzyme activity, but some residual activity remained.

The effect of native and heat inactivated Eno1 on glucose uptake wascompared in human skeletal muscle myotubes following the methodsdescribed above. As shown in FIG. 3B, myotubes treated with native(active) Eno1 exhibited significantly higher glucose uptake relative tomyotubes that were not treated with Eno1. Myotubes treated with heatinactivated Eno1 exhibited significantly lower glucose uptake comparedto myotubes treated with active Eno1. These results indicate that theeffect of Eno1 on glucose uptake is dependent on Eno1 enzyme activity.The increase in glucose uptake observed in the heat inactivated Eno1relative to the control containing no Eno1 was likely due to theresidual Eno1 activity of the heat inactivated enzyme.

Example 3 Mouse Models of Diet Induced Obesity (DIO) Mice

Two essentially equivalent models of diet induced obesity were used inthe methods provided herein.

In the first method, male C57BL/6J mice were obtained from JacksonLaboratories (Bar Harbor, Me.) and initially housed 4-5 per cage at 22°C. on a 12:12 hr day-night cycle. Beginning at 6 weeks of age, mice werefed with a high-fat diet (Research Diets Cat #: D12492; 60 kcal % fat,20 kcal % protein, and 20 kcal % carbohydrate). Lean control mice werealso obtained and fed a standard diet. Body weight of DIO mice beforeexperiments was significantly heavier than that of lean control mice. Inone study, DIO mice weighed 38.4±0.6 g whereas lean mice weighed29.9±0.5 g (p<0.05).

In the second method, diet induced obese male C57BL/6J mice (12 weekold) and control lean mice (12 week old) were obtained from JacksonLaboratories (Bar Harbor, Me.) and initially housed 4-5 per cage at 22°C. on a 12:12 hr day-night cycle. Mice were acclimated in animalfacility for one week before treatments and maintained with a high-fatdiet for DIO group (Research Diets Cat #: D12492; 60 kcal % fat, 20 kcal% protein, and 20 kcal % carbohydrate) or a low fat diet (10% kcal %fat) for lean group.

Example 4 Treatment of Glucose Intolerance with Eno1 in Diet InducedObesity (DIO) Mice

The experimental protocol was started when the mice (n=10 per group)were obese after being maintained for 7 weeks on a high fat diet.Osmotic minipumps (Model 1004, Alzet, Cupertino, Calif.) were filledfollowing manufacturer's guidelines with 0.1 ml of the Eno1 peptide orvehicle (phosphate buffered saline (PBS), pH 7.0). The pumps were primedin sterile saline at 4° C. overnight. Mice were anesthetized withisoflurane (1-3% in 100% oxygen) and scrubbed with 70% isopropanol andbetadine solutions before surgery. A small subcutaneous incision wasmade in the midscapular region, the pump was inserted and the wound wassutured. Animals were allowed to recover before returning to their homecages. The implantation of the subcutaneous osmotic minipumpscontinuously infused peptide at a constant rate of 0.11 μl/hr for fourweeks. Pump-exchange surgeries were performed every 4 weeks. Thepurified Eno1 treatment doses calculated by pump infusion rate was 10μg/kg body weight.

Glucose tolerance tests (GTT) were performed after 6 h of fasting usingroutine methods. Briefly, initial fasting blood glucose levels weredetermined, followed by intraperitoneal (ip) injection of 20% dextrosesolution at a dose of 1.5 g/kg body weight. Blood glucose levels weremeasured from the tail vein at 15, 30, 60, 90, and 120 minutes after theglucose injection using an ACCU-CHEK® Advantage glucometer (ROCHE®Diagnostics, Indianapolis, Ind.). The area under the curve (AUC) duringthe GTT was calculated with Graphpad Prism® software, and studentt-tests were performed for significance between different treatmentgroups. The results are shown in FIGS. 4A and 4B.

As can readily be observed, mice treated with Eno1 had a significantdecrease in blood glucose area under the curve as compared to untreatedmice (p=0.017). These data demonstrate that treatment of obese mice withEno1 protein increases glucose tolerance as demonstrated by a glucosetolerance test and indicate that Eno1 is effective in the treatment ofinsulin resistance, glucose intolerance, and type 2 diabetes.

Example 5 Generation of a PAMAM Dendrimer, Muscle Targeted Eno1

Having demonstrated the efficacy of Eno1 in increasing glucose uptake inmyotubes and increasing glucose tolerance upon systemic administration,a muscle targeted Eno1 was generated to analyze its efficacy inincreasing glucose tolerance. Detectably labeled G5-PAMAM dendrimerscontaining the muscle targeting peptide (MTP) ASSLNIA and/or Eno1 weregenerated using the methods described below. A range of different ratiosof MTP to dendrimer were evaluated, including MTP containing dendrimerswhich contained about 10 MTP peptides per dendrimer, about 3 MTPpeptides per dendrimer, or about 1 MTP peptide per dendrimer.

The process of preparing Eno1 dendrimer complexes includes theidentification of optimal ratios and concentrations of the reagents.Stock solutions of Eno1 were prepared in buffer and the protein solutionwas mixed with G5 dendrimer-muscle targeting peptide (MTP) conjugate indifferent ratios. A range of different ratios of dendrimer to Eno1 werealso evaluated, including Eno1 containing dendrimers which containedabout one dendrimer per molecule of Eno1 protein or about fivedendrimers per molecule of Eno1 protein.

The stability of the Eno1-dendrimer-SMTP complex was evaluated atdifferent temperatures, and stability was determined over a 3-4 monthtime period by measuring Eno1 activity using a commercially availableEno1 assay. The selected conjugates were also evaluated usingbiophysical techniques, including Dynamic Light Scattering (DLS) andUV-Vis spectroscopy to confirm complexation between thedendrimer-peptide conjugate and Eno1.

Determination of the Purity of Eno1:

The purity of a 5.32 mg/mL solution of Eno1 protein was checked byCoomassie and Silver staining and Western blotting. Several dilutions ofthe Eno1 protein ranging from 10 μg/well to 100 ng/well were preparedand loaded on a 12-well, 4-12% mini-PROTEAN® TGX gel [BIO-RADCat#456-1095 Lot#4000 79200]. The lane assignments were as follows; Lane1: Ladder (Precision Plus Protein Standard Dual Color [BIO-RADCat#161-0374]; Lane 2: Eno1 (10.0 μg); Lane 3: Eno1 (1.0 μg); Lane 4:Eno1 (0.1 μg); Lane 5: Ladder (Precision Plus Protein Standard DualColor [BIO-RAD Cat#161-0374]; Lane 6: Eno1 (10.0 μg); Lane 7: Eno1 (1.0μg); Lane 8: Eno1 (0.1 μg); Lane 9: Ladder (Precision Plus ProteinStandard Dual Color [BIO-RAD Cat#161-0374]; Lane 10: Eno1 (10.0 μg);Lane 11: Eno1 (1.0 μg); Lane 12: Eno1 (0.1 μg). The SDS-PAGE was run at200 V for 20-25 min.

Coomassie Staining:

After the gel was run, the gel was split into 3 equal parts. One of theparts was stained with Coomassie Stain. Briefly, the gel was soaked in100 mL of Coomassie Stain solution (0.025% Coomassie Stain in 40%Methanol and 7% Acetic Acid) and heated for one minute in a microwave.Then the gel was left to stain with gentle agitation for 45 minutes.After the staining was complete, the gel was destained using destainingsolution (40% Methanol and 7% Acetic Acid) until the background stainingwas acceptable.

As shown in FIG. 5, the protein ran as a single band of about 47 KDa,which is consistent with the size of Eno1.

Silver Staining:

Since Coomassie Staining is not a sensitive method for visualization ofthe protein bands, another portion of the gel was stained with SilverStain using BIO-RAD's Silver Staining Kit [BIO-RAD Cat#161-0443]. TheModified Silver Stain Protocol was followed.

As shown in FIG. 6, extra bands can be seen in each lane, whichcorrespond to the bands of the ladder. This is due to the leakage of theladder into the neighboring lanes. The three bands marked with an arroware not from the ladder. The most prominent band is about 47 kDa, whichis consistent with the size of Eno1. There are two extra bands in thepurified protein but these bands are faint, indicating that overallpurity of the Eno1 was relatively high.

Western Blot Analysis:

The identity of Eno1 was further confirmed by Western blot. For thispurpose, the final portion of the gel was transferred into 100 mL ofTris-Glycine buffer and transferred onto 0.2 μm PVDF membrane (BIO-RAD)using a transblot SD semi-dry transfer apparatus (BIO-RAD) at 20 V for2.0 h. The efficiency of the transfer was checked by observing thepresence of the pre-stained ladder bands on the membrane. The membranewas dried for 1.0 h. The membrane was then wetted with methanol for 1.0min and blocked with 15.0 mL ODYSSEY® Blocking Buffer (LICOR) at roomtemperature for 2.0 h.

After the blocking was complete, the membrane was incubated with 15.0 mLODYSSEY® Blocking Buffer containing 30 μL of anti-ENOA-1 m-Ab (mouse)(purchased from ABNOVA) overnight at 4° C. Then the membrane was washedwith 3×30 mL of 1×PBS-T with shaking for 5 minutes each. The membranewas incubated with 15.0 mL ODYSSEY® Blocking Buffer containing 5 μL ofGoat anti-mouse secondary antibody labeled with IRDye® 800CW (purchasedfrom LICOR) for 2.0 h at room temperature. After the incubation, themembrane was washed with 3×30 mL of 1×PBS-T followed by 2×30 mL of 1×PBSwith shaking for 5 minutes each. Finally, the membrane was imaged usingthe LICOR ODYSSEY Infrared Imager. As shown in FIG. 7, Western Blotanalysis confirmed that the dominant band at 47 kDa was Eno1.

Zeta (ζ)-Potential Characterization of Enolase-I/G5-PAMAM-SMTP:

Eno1 and Generation 5 PAMAM dendrimers decorated with 2-3 SkeletalMuscle Targeting Peptides (SMTPs) were complexed at varied ratios toform Eno1/G5-SMTP protein/dendrimer complexes. The concentration of thedendrimer was kept constant at 1.0 μM and the Eno1 concentration wasvaried between 0.1 μM-10.0 μM. Table 2 below describes how theEnolase-I/G5-dendrimer/SMTP mixtures were prepared.

TABLE 2 Various combinations of Eno1 and G5-dendrimer/SMTP for formationof dendrimer complexes. G5-Dendrimer Eno1/Dendrimer Eno1 SMTP PBS bufferMolar Ratio (5.32 mg/mL) (30.0 mg/mL) pH = 7.40 10:1  88.3 μL 1.03 μL910.67 μL 5:1 44.15 μL  1.03 μL 954.82 μL 2:1 17.66 μL  1.03 μL 981.31μL 1:1 8.83 μL 1.03 μL 990.14 μL 1:2 4.42 μL 1.03 μL 994.55 μL 1:5 1.77μL 1.03 μL  997.2 μL  1:10 0.88 μL 1.03 μL 998.09 μL

Each sample was prepared by adding G5-dendrimer/SMTP to the respectiveamount of PBS. Enolase was then added to the G5-dendrimer/SMTP solutionin a drop wise fashion while vortexing at low speed. The sample was thenincubated at room temperature for 20 minutes prior to analysis.

Size measurements were made using the Zetasizer Nano Z90s instrumentfrom Malvern Instruments. The default parameters were used for themeasurements and three separate measurements of each sample werecollected. FIG. 8 shows representative Zeta (ζ)-Potential data for threesamples of Eno1/G5-dendrimer/SMTP complexes having a 2:1 molar ratio ofEno1 to dendrimer/SMTP. Zeta (ζ)-Potential was measured using DynamicLight Scattering. As shown in FIG. 8, the peaks of the three samples arematching, indicating a uniform charge distribution of the Enolase-SMTPdendrimer complex.

Stability of Enolase-I/G5-SMTP Complexes:

The stability of the Enolase-I/G5-dendrimer/SMTP conjugates was measuredby using the ENO1 Human Activity Assay Kit (ABCAM, Cambridge, Mass.;Catalogue No. ab117994). Briefly, the sample was added to a microplatecontaining a monoclonal mouse antibody specific to Eno1. The microplatewas incubated at room temperature for 2 hours, and Eno1 wasimmunocaptured within the wells of the microplate. The wells of themicroplate were washed to remove all other enzymes. Eno1 activity wasdetermined by following the consumption of NADH in an assay buffer thatincluded pyruvate kinase (PK), lactate dehydrogenase (LDH) and therequired substrates 2-phospho-D-glycerate (2PG) and NADH. Eno1 converts2PG to phosphoenolpyruvate, which is converted to pyruvate by PK.Pyruvate is converted to lactate by LDH, and this reaction requiresNADH. The consumption of NADH was monitored as decrease of absorbance at340 nm.

The activity of Enolase-I/G5-dendrimer/SMTP conjugates that were storedat different temperatures at different time points was measured usingthe assay described above. A concentration of 500 ng of Eno1 wasselected for testing because this concentration falls in the middle ofthe dynamic range of the assay kit. Two different sets of solutions wereprepared. One set (control) contained Eno1 alone (i.e. unconjugatedEno1) and the other set contained Eno1/G5-dendrimer/SMTP mixtures. Thesemixtures were then kept at −80° C., −20° C., 4° C., 22° C., and 37° C.The results showed that in the first week all of the samples wereactive, and the Eno1/G5-dendrimer/SMTP conjugates seemed to have aslightly higher activity than Eno1 alone. However, the activities of thesolutions, regardless of whether or not they contained dendrimers,steadily decreased in the next two weeks. By week 3, the solutions thatwere stored at 4° C., 22° C., and 37° C. showed no activity, while thesolutions that were stored at −80° C., and −20° C. showed significantstability. At the end of the study (Week 10), The Eno1/G5-dendrimer/SMTPsolution that was kept at −80° C. retained about 90% of its activitywhereas Eno1 alone was only 35% active. On the other hand,Eno1/G5-dendrimer/SMTP solution that was kept at −20° C. was about 24%active, whereas Eno1 alone stored at −20° C. was not active (FIG. 9).

Example 6 In Vivo Eno1 Targeting Studies with G5 PAMAM Dendrimers

A detectably labeled PAMAM dendrimer complex containing Eno1 wasprepared using the method provided in the prior example and analyzed fortissue distribution in mice after subcutaneous injection. Specifically,for 72 hours prior to injection mice were fed alfalfa free food to limitbackground fluorescence. Mice were injected with 3 μg ENO1/mousesubcutaneously 150 μl total (75 μl left laterally, 75 μl rightlaterally). The molar ratio of dendrimer to Eno1 in the complex was 5:1.One, 4, and 24 hours post injection animals were sacrificed, skinned,and organs removed in preparation for LI-COR imaging. The results areshown in FIG. 10A.

As shown, at 1 hour, general systemic distribution of the Eno1-PAMAMdendrimer was observed. After 4 hours, significant accumulation of theEno1-PAMAM dendrimer was observed in liver, kidney, and subcutaneousfat, as well as in the upper torso. After 24 hours, the Eno1-dendrimercomplex was substantially cleared and observed substantially in theliver and kidney.

A follow-up study was performed using the skeletal muscle targetedEno1-PAMAM dendrimer complex containing the SMTP “ASSLNIA”. A detectablylabeled PAMAM dendrimer complex containing Eno1 and SMTP ((Enolase-VivoTag680x1)-(G5-SMTP)) was prepared using the method provided in the priorexample. The molar ratio of dendrimer to SMTP in the complex was 1:1.The experiments were performed essentially as described above. Theskeletal muscle targeted Eno1-PAMAM dendrimer complex was administeredat a dose of 50 μg/kg body weight. These images in FIG. 10B were takenafter 1 hr of injection. Organs, other than the heart, were retained inthe body. As can be readily observed, the muscle-targeted Eno1 dendrimercomplex was targeted to skeletal muscle, not heart. These resultsdemonstrate that the skeletal muscle targeted Eno1-PAMAM dendrimercomplex can be used for the delivery of Eno1 to skeletal muscle cells.

Example 7 Treatment of Glucose Intolerance with Muscle Targeted Eno1 inDiet Induced Obesity (DIO) Mice

Diet induced obese male C57BL/6J mice (12 week old) and control leanmice (12 week old) were obtained from Jackson Laboratories (Bar Harbor,Me.) and initially housed 4-5 per cage at 22° C. on a 12:12 hr day-nightcycle. Mice were acclimated in animal facility for one week beforetreatments and maintained with a high-fat diet for DIO group (ResearchDiets Cat #: D12492; 60 kcal % fat, 20 kcal % protein, and 20 kcal %carbohydrate) or a low fat diet (10% kcal % fat) for lean group.

Beginning at 13 weeks of age, all mice received daily subcutaneousinjections of either saline or different complexes with combinations ofG5 dendrimer, skeletal muscle targeting peptide (SMTP), and purifiedEno1 (50 μg/kg body weight) for duration of 4 weeks. During the 4 weeksof the treatment portion of the experiment, intraperitoneal glucosetolerance tests (IPGTT) were performed weekly. Body weight, fed glucose,and fasted glucose were measured weekly during treatment period. Thetreatment groups are shown below:

1. LFD—Lean Controls—no injection

2. HFD—saline control (volume equivalent to G5+SMTP+Eno1)

3. HFD—G5 only (equivalent to 50 μg/kg of G5+SMTP+Eno1)

4. HFD—G5+SMTP (equivalent to 50 μg/kg of G5+SMTP+Eno1)

5. HFD—G5+Eno1 (50 μg/kg body weight)

6. HFD—G5+SMTP+Eno1 (50 μg/kg body weight)

The molar ratio of dendrimer to Eno1 in the complexes was 5:1, the molarratio of dendrimer to SMTP in the complexes was 1:1, and the dendrimerwas acetylated. Results from the study are provided in FIGS. 11, 12, 13,14 and 15.

In this small cohort, none of the treatment regimens were found to havea significant effect on body weight in the DIO mice at any time duringthe study (see FIG. 11).

Treatment of mice with a single dose of the dendrimer bound muscletargeted Eno1 was demonstrated to have an effect on blood glucose levelsat the earliest time points tested. As shown in FIG. 12, one hour afteradministration of 50 μg/kg of G5+SMTP+Eno1, a reduction of blood glucosewas observed as compared to a saline control, with the maximum reductionobserved at 4 hours. The effect was no longer observed at 24 hours afterthe single injection.

At one week after initiation of administration of the dendrimer boundmuscle targeted Eno1, glucose tolerance in the DIO mice treated with theEno1 dendrimer SMTP complex (DIO Enolase-1+NP+SMTP) were significantlylower than glucose tolerance in DIO mice treated with the dendrimer SMTPcomplex alone (DIO NP+SMTP) (see FIGS. 13A and 13B).

At two weeks after initiation of administration of the dendrimer boundmuscle targeted Eno1, glucose tolerance in the DIO mice was stillsignificantly improved (see FIGS. 6C and 6D). The improvement of glucosetolerance was dependent on the presence of Eno1 in the dendrimer complex(DIO G5+SMTP vs. DIO Eno1 G5+SMTP, p=5.7×10⁻⁵, p=0.002). The effect wasno longer observed 23 hours after the single injection (data not shown).

The beneficial effect of G5+SMTP+Eno1 treatment observed at weeks 1 and2 was sustained through week 4 (see FIGS. 14A and 14B). Specificallyglucose tolerance in the DIO Eno1 G5+SMTP treated mice was similar tothat in lean mice. The improvement of glucose tolerance was significantand dependent on the presence of Eno1 in the dendrimer complex (DIOG5+SMTP vs. DIO Eno1 G5+SMTP, p=0.0017). The effect was no longerobserved 23 hours after the single injection (data not shown).

These results show that dendrimer bound, muscle targeted Eno1 iseffective in increasing glucose tolerance in a model of diet inducedobesity, and that G5+SMTP+Eno1 is effective in normalizing blood glucosein a mouse model of diet induced obesity. These results demonstrate thatEno1 is useful in the treatment of elevated blood glucose, glucoseintolerance, and diabetes, particularly type 2 diabetes.

The mice were treated as described above for an additional 4 weeks (8weeks treatment in total), and serum lactate levels were determined inlean mice, diet induced obesity (DIO) mice, DIO mice treated withG5-dendrimer, and DIO mice treated with Eno1/G5-dendrimer/SMTP complexafter 8 weeks of treatment. Lactate levels in serum were measured usinga lactate colorimetric assay kit from Biovision (Milpitas, Calif.). Asshown in FIG. 15, Eno1/G5-dendrimer/SMTP complex significantly reducedlactate serum levels. This result suggests that the reduced glucoselevels observed in the DIO mice treated with the Eno1/G5-dendrimer/SMTPcomplex is due to increased glucose oxidation, rather than shunting ofglycolysis to lactate. This would minimize the undesirable effects oflactate acidosis.

Example 8 Treatment of Glucose Intolerance with Muscle Targeted Eno1 ina Genetic Model of Obesity, Db/Db Mice

Male obese and diabetic db/db mice (male BKS.Cg-m+/+Lepr^(db)/J) micewere obtained from a commercial vendor. All mice were housed 2-3 percage at 22° C. on a 12:12 hr day-night cycle and are acclimated for 3weeks at animal facility on a standard chow diet. At 8 weeks of age, thefollowing subcutaneous injections of either saline or differentcomplexes with combinations of G5 dendrimer, skeletal muscle targetingpeptide (SMTP), and purified Eno1 were administered once daily bysubcutaneous administration (n=6 per group). The treatment groups are asfollows:

1. db/db with saline injection

2. db/db with G5+SMTP (volume equivalent to Eno1+G5+SMTP at 25 ug/kgdose)

3. db/db with Eno1 (25 ug/kg body weight)+G5+SMTP

4. db/db with Eno1 (50 ug/kg body weight)+G5+SMTP

The molar ratio of dendrimer to Eno1 in the complexes was 5:1, and themolar ratio of dendrimer to SMTP in the complexes was 1:1, and thedendrimer was acetylated.

At day 7, the mice were administered the appropriate agent and returnedto the cage for 6 hours without food prior to administration of an IPGTTas described in the Example above. The results are shown in FIGS. 16Aand 16B. As can be readily observed, treatment of mice with Eno1+G5+SMTPresulted in an increase in glucose tolerance after glucose challengewith a significant increase in glucose clearance observed in the micetreated with Eno1 (50 ug/kg body weight)+G5+SMTP as compared to the micetreated with G5+SMTP (p=0.015).

The study was continued with three out of the six mice in each of thetreatment groups listed above. Mice were administered the indicatedagent for an additional week (2 weeks total). The effect of Eno1 onlowering fed blood glucose was tested. Specifically, without controllingthe intake of food, blood glucose levels in mice were assessed for twohours immediately after administration of the active agent. The resultsare shown in FIGS. 17A and 17B. As shown, administration of Eno1 (50ug/kg body weight)+G5+SMTP was demonstrated to decrease fed bloodglucose and resulted in a statistically significant reduction in bloodglucose 30 minutes after administration as compared to administration ofG5+SMTP. However, the reduced blood glucose observed 30 minutes afterEno1+G5+SMTP treatment were not maintained at 24 hours after Eno1injection (FIG. 18).

Accordingly, the effect of twice daily dosing of Eno1+G5+SMTP on bloodglucose levels was also evaluated in the db/db mice. Treatments wereadministered by subcutaneous injection twice daily, once in the morningand once in the evening, for four weeks. The treatment groups were asfollows:

1. PBS

2. 100 μg/kg body weight Eno1+G5+SMTP

3. 200 μg/kg body weight Eno1+G5+SMTP

The molar ratio of dendrimer to Eno1 in the Eno1+G5+SMTP complex was5:1, and the molar ratio of dendrimer to SMTP in the Eno1+G5+SMTPcomplex was 1:1, and the dendrimer was acetylated.

The total daily dose for treatment group 2 was 200 μg/kg body weightEno1+G5+SMTP and the total daily dose for treatment group 3 was 400μg/kg body weight Eno1+G5+SMTP. Without controlling the intake of food,fed blood glucose levels were assessed in the mice 16 hours after theevening injection (i.e. before the morning injection). As shown in FIG.19, twice daily injection of 200 μg/kg body weight Eno1+G5+SMTPdecreased fed blood glucose levels relative to the control PBStreatment.

Thus, treatment of mice with Eno1 G5+SMTP was shown to normalize glucoseresponse in db/db mice. The data described in Examples 7 and 8 togetherdemonstrate that Eno1 is effective in increasing glucose tolerance inboth an induced and a genetic model of type 2 diabetes.

Example 9 Comparative Toxicity of Acylated vs. Non-Acylated SMTPContaining Dendrimers

The toxicity of acylated and non-acylated dendrimers containing SMTPwere compared using creatine kinase and caspase 3 assays. Mice wereinjected with one of staurosporine (positive control),staurosporine+inhibitor (negative control); G5 PAMAM dendrimers, SMTP-G5PAMAM dendrimers, and acylated SMTP-G5 PAMAM dendrimers. The molar ratioof dendrimer to Eno1 in the complexes was 5:1, and the molar ratio ofdendrimer to SMTP in the complexes was 1:1. After injection samples werecollected and assayed for the creatine kinase levels as a percent oftotal cell lysate and caspase 3 activity using commercially availablekits. The results are shown in FIG. 20. As shown in FIG. 20,administration of the G5 PAMAM dendrimers at both 1 uM and 3 uMconcentrations and administration of SMTP-G5 PAMAM dendrimers at 3 uMconcentration resulted in a significant increase in creatine kinaseactivity. No such effect was observed with the acylated SMTP-G5 PAMAMdendrimers. Similarly, a significant increase in caspase 3 activity wasobserved after administration of 3 uM G5 PAMAM dendrimers and SMTP-G5PAMAM dendrimers. However, no increase in caspase 3 activity wasobserved upon administration of the acylated SMTP-G5 PAMAM dendrimers.These results demonstrate that acylation of SMTP-G5 PAMAM dendrimersreduces toxicity.

Example 10 Treatment of Glucose Intolerance with Muscle Targeted Eno1 ina Genetic and Induced Models of Type 2 and Type 1 Diabetes

Male obese mice, diabetic db/db mice (male BKS.Cg-m+1+Lepr^(db)/J), NOD1mice, or streptazocin treated mice are obtained or generated. All miceare housed 2-3 per cage at 22° C. on a 12:12 hr day-night cycle and areacclimated for at least 1 week at animal facility on an appropriate chowdiet (i.e., high fat diet for obese mice, normal chow for other mice).At an appropriate age, typically about 8 weeks of age, subcutaneousinjections of either saline or different complexes with combinations ofG5 dendrimer, skeletal muscle targeting peptide (SMTP), and purifiedEno1 (25 or 50 μg/kg body weight) are administered daily for duration of1-2 weeks. Implantable pumps (e.g., ALZET pumps) as described above canbe used for administration on a daily or continuous basis.Alternatively, the agents can be administered intramuscularly in variousformulations. Intramuscular injections are typically performed on a lessfrequent basis than subcutaneous injections (e.g., typically about onceper week).

During the 2 weeks of time-course, intraperitoneal glucose tolerancetests (IPGTT) are performed and fasting and fed blood glucose ismonitored, either randomly or in a time course after administration ofthe agent. Body weight is measured weekly during treatment period. Thetreatment groups include at least one control (e.g., 1 or 2) and atleast one Eno1 treatment from the list shown below:

1. Saline injection

2. G5+SMTP (volume equivalent to Eno1+G5+SMTP at 25 ug/kg bodyweight/day)

3. Eno1 (25 ug/kg body weight/day)+G5+SMTP

4. Eno1 (50 ug/kg body weight/day)+G5+SMTP

5. Eno1 (25 ug/kg body weight/day)

6. Eno1 (50 ug/kg body weight/day)

Dosages provided are exemplary and are not to be considered limiting.

Treatment of mice with Eno1 G5+SMTP is demonstrated to normalize glucoseresponse in the diabetic mice.

Example 11 Assessment of Glucose Levels and Glucose Response in Mice

The intraperitoneal glucose tolerance test (IPGTT) is described aboveand routinely used to assess glucose tolerance and insulin response.Other exemplary methods that can be used to confirm the efficacy of Eno1in normalizing blood glucose and insulin response are provided below.Methods to assess body composition and metabolism are also providedbelow.

Intraperitoneal Insulin Tolerance Test (IPITT)

Insulin tolerance test (ITT) is performed after 1 hour fasting to assesspyruvate metabolism. Initial blood glucose levels is determined,followed by injection (ip) of human insulin (1-2 U/kg; Humulin R; EliLilly, Indianapolis, Ind.). Blood glucose levels are measured from thetail vein as described above at 15, 30, 60, 90, and 120 min after theinsulin injection. The insulin injection amount is determinedempirically by insulin response due to the onset of the hepatic insulinresistance in the mice subjected to the high fat diet.

Intraperitoneal Pyruvate Tolerance Test (IPPTT)

Pyruvate challenge test is administered after 6 h of fasting. Initialblood glucose levels are determined, followed by injection (ip) ofpyruvate dissolved in saline (2 g/kg; Sigma, St. Louis, Mo.). Bloodglucose levels are measured from the tail vein as described above at 15,30, 60, 90, and 120 min after the pyruvate injection. The area under thecurve (AUC) during the test is calculated.

Fed Blood Glucose Levels

Blood samples are obtained from mice fed ad libitum either randomly orat a defined time or time interval after administration of an agent ofinterest. Blood glucose levels are measured.

Fasting Blood Glucose Levels

Blood samples are obtained from mice after a fast of a predefined timeperiod (typically about 6-8 hours) at a defined time or time intervalafter administration of an agent of interest. Blood glucose levels aremeasured.

Assessment of Indicator Levels to Assess Blood Glucose Levels

Mouse models of type 1 or type 2 diabetes are treated with one or moreagents of the invention and appropriate controls. Levels of HbA1c and/orEno1 protein and/or RNA are monitored to determine blood glucose levelsover a sustained period.

Dual-Energy X-Ray Absorptiometry (DEXA)

The body mass composition of different treatment groups is determined bydual-energy x-ray absorptiometry (DEXA) scanning using LUNAR PIXImus®mouse densitometer following the procedures recommended by themanufacturer. Lean body mass, fat body mass, total body tissue weight,bone density, and bone mineral content are recorded and analyzed.

Comprehensive Lab Animal Monitoring System (CLAMS)

The CLAMS (Columbus Instruments, Columbus, Ohio, USA) metabolicmonitoring cages are used to simultaneously monitor horizontal andvertical activity, feeding and drinking, oxygen consumption, and CO₂production. ASO injected and control mice are individually placed inCLAMS cages and monitored over a 4-day period after acclimation to thecages for 1-2 days. The various parameters are recorded in both fastedand fed conditions. Food and water consumption are measured directly asaccumulated data. Hourly files display all measurements for eachparameter: volume of oxygen consumed, ml/kg per h (VO₂), volume ofcarbon dioxide produced, ml/kg per h (VCO₂), respiratory exchange ratio,heat (kcal/h), accumulated food (g), accumulated drink (g), XY totalactivity (all horizontal beam breaks in counts), XY ambulatory activity(minimum three different, consecutive horizontal beam breaks in counts),and Z activity (all vertical beam breaks in counts). The data arerecorded during the 30-s sampling period. The CLAMS data are analyzed bynormalizing with lean body mass.

Example 12 Effect of Eno1 on Insulin Stimulated p-Akt in Human SkeletalMuscle Myotubes

The effect of purified Eno-1 on insulin stimulated p-Akt (S473) proteinlevels was determined in cell cultures of human skeletal muscle myotubeswith our without insulin treatment. p-Akt protein levels were measuredby ELISA. As shown in FIG. 21, insulin treatment increased p-Akt proteinlevels in the absence of Eno1 treatment, and the effect of insulin onp-Akt protein levels was similar with or without Eno1 treatment. Theseresults indicate that Eno1 does not influence insulin stimulation ofp-Akt protein levels, suggesting that the effects of Eno1 on glucoseuptake in muscle cells are independent of insulin and would occur incells exhibiting insulin resistance.

Example 13 Eno1 is Associated with Increased Glucose Flux in HumanSkeletal Muscle Myotubes

Glucose transporter 1 (Glut1) and Glucose transporter 4 (Glut4) areinvolved in the transport of glucose across the plasma membrane and arethe predominant facilitative glucose transporters within skeletal muscle(Jones et al., 1998, Journal of Applied Physiology, Vol. 84, pp.1661-1666). Glut4 is responsible for insulin-regulated glucose transportinto the cell. Myogenin is a muscle specific transcription factor thatmay be involved in regulating Glut1 and Glut4 expression (see Jones etal., above). Hexokinase 2 (HK2) phosphorylates glucose to formglucose-6-phosphate (G6P) and is the predominant hexokinase in skeletalmuscle.

Expression of Glut1, Glut 4, HK2 and myogenin was measured in cellcultures of human skeletal muscle myotubes with or without Eno1treatment. The myotubes were treated with purified Eno1 which wasprepared as described in Example 2. Glut1, Glut4, HK2 and myogenin mRNAlevels were determined by quantitative PCR. Glut1 protein levels weredetermined by MS proteomics analysis. As shown in FIGS. 22A and 22B,Eno1 treatment increased Glut1, Glut4 and HK2 mRNA levels, and Glut1protein levels. Because these proteins are involved in glucose transportand metabolism, these results indicate that Eno1 treatment is associatedwith increased glucose flux in skeletal muscle.

To further investigate the role of Eno1 in glucose flux, G6P andphosphoenol pyruvate (PEP) levels were measured in glucose starved andglucose stimulated human skeletal muscle myotubes with or withouttreatment with purified Eno1. Glucose starving was performed byincubating the myotubes in glucose free DMEM for 15 min. Glucosestimulation was performed by treating the myotubes with 5 mM glucose for15 min. G6P and PEP levels were measured using assay kits from Biovision(Milpitas, Calif.; Cat. Nos. K657-100 and K365-100). As shown in FIGS.23 and 24, Eno1 treatment increased G6P and PEP levels in both glucosestarved and glucose stimulated human skeletal muscle myotubes, furtherindicating that Eno1 treatment is associated with increased glucose fluxin skeletal muscle.

Example 14 Eno1 Mode of Action

To further investigate the mode of action of Eno1 in glucose uptake, theoxygen consumption rate (OCR) and extracellular acidification rate(ECAR) were measured in human skeletal muscle myotube cells. OCR is anindicator of mitochondrial respiration and ECAR is an indicator ofglycolysis.

For OCR experiments, various compounds were added sequentially to thecells to induce changes in OCR. For example palmitate and carbonylcyanide m-chlorophenylhydrazone (CCCP, an uncoupler of oxidativephosphorylation) were added to increase OCR and etomoxir (a fatty acidoxidation inhibitor) was added to decrease OCR. KHB buffer (pH 7.4) wasadded to each well and measurements were performed every 3 min with 2min intermeasurement mixing. BSA-conjugated palmitate (finalconcentration 200 mmol/L), CCCP (final concentration 2 μM) and etomoxir(final concentration 50 mmol/L) were injected sequentially. As shown inFIG. 25, Eno1 treatment increased OCR. These results indicate that Eno1treatment is associated with increased mitochondrial free fatty acidoxidation in human skeletal muscle myotubes.

For ECAR experiments with glucose as a substrate, sodium carbonate andglucose/pyruvate-free DMEM were used. Glucose, oligomycin and 2-DG wereinjected sequentially to give final concentrations of 25 mmol/L. Asshown in FIG. 26, Eno1 treatment increased ECAR, indicating that Eno1treatment is associated with increased glycolytic activity and capacity.

To determine the mitochondrial content of human skeletal muscle myotubestreated with Eno1, myotubes were treated with 500 ug/ml or 1000 μg/mlEno1 for 48 hours and then Mitotracker green (Invitrogen), a greenfluorescent mitochondrial stain, was added. After 15 min of staining,the myotubes were trypsinized, washed, and subjected to flow cytometryto determine mitochondrial content. As shown in FIG. 27A, Eno1 treatmentdoes not influence mitochondrial content.

Mitochondrial ROS was also detected in the Eno1 treated human skeletalmuscle myotubes described above. Mitochondrial ROS was determined bytreating cells with Dihydrorhodamin 123 (Life Technologies), anuncharged and nonfluorescent reactive oxygen species (ROS) indicatorthat can passively diffuse across membranes where it is oxidized tocationic rhodamine 123 which localizes in the mitochondria and exhibitsgreen fluorescence. The myotubes were then trypsinized, washed, andsubjected to flow cytometry. Treatment of human skeletal muscle myotubeswith Eno1 did not affect mitochondrial reactive oxygen speciesproduction (FIG. 27B).

These results indicate that the mode of action of Eno1 is not due tochanges in mitochondrial content or ROS production.

To further investigate the mode of action for Eno1, non-phosphorylated5′ AMP activated protein kinase (AMPK) and phosphorylated AMPK (pAMPK)levels were measured in basal and serum starved human skeletal musclemyotubes treated with 0, 500, or 1000 μg/ml Eno1. Basal human skeletalmuscle myotubes were treated with normal differentiation mediumcontaining 2% horse serum, while serum-starved myotubes were starvedwith serum free DMEM containing 0.5% BSA for 3 hours before lysis of themyotubes. AMPK and pAMPK levels were determined by Western blot usingantibodies specific to the phosphorylated or non-phosphorylated form ofthe kinase. An antibody specific to Lamin A/C was used to confirm evenloading among samples. As shown in FIGS. 28A and 28B, Eno1 treatment didnot affect pAMPK levels in basal or serum starved myotubes. AMPKactivation or phosphorylation is one of the major insulin independentpathways that regulate glucose uptake in skeletal muscle, for exampleduring muscle contraction. Accordingly, the lack of an effect of Eno1 onpAMPK levels suggest a novel mode of action for Eno1 beyond conventionalsignal transduction.

Example 15 Eno1 Binding Partners in Human Skeletal Muscle Myotubes

To further investigate the mode of action for Eno1, the binding partnersof Eno1 were compared in untreated human skeletal muscle myotubes(containing endogenous Eno1) and human skeletal muscle myotubes treatedwith 50 μg/ml or 100 μg/ml of 6× Histidine tagged exogenous Eno1.Endogenous Eno1 was immunoprecipitated in the untreated myotubes usingan antibody specific to Eno1. The exogenous Eno1 was immunoprecipitatedusing the 6× Histidine Tag antibody. The binding partners of endogenousEno1 and exogenous Eno1 were identified by quantitative proteomics, andthe identity of the binding partners was confirmed by Western blotand/or reverse immunoprecipitation.

Nicotinamide phosphoribosyltransferase (Nampt) was identified as abinding partner of both endogenous and exogenous Eno1. Nampt catalyzesthe synthesis of nicotinamide mononucleotide (NMN) from nicotinamide andis involved in muscle contraction and secretion. Eno1 may interact withNampt as part of the glycolysis complex, as depicted in FIG. 29.

Example 16 Interaction of Eno1 and Nampt

Nampt activity was determined in human skeletal muscle myotubes treatedwith 500 ug/ml or 1000 μg/ml Eno1 in differentiation medium for 48 hoursafter 4 days of differentiation. Myotube lysates were subjected toimmunoprecipitation using either IgG or anti-Nampt antibody (CloneAF-1E12) from Cyclex (Nagano, Japan). The immunoprecipitated myotubelysates were subjected to Nampt activity assay using Cyclex Namptactivity assay kit (#CY-1251). As shown in FIG. 30, Eno1 treatmentincreased Nampt activity. Eno1 treatment also increased secretion ofNampt (eNampt) in human skeletal muscle myotubes (data not shown).

2-DG uptake was measured in human skeletal muscle myotubes which hadbeen serum starved for 3 hours and then treated with recombinantextracellular Nampt (eNampt) from Abcam (Cambridge, Mass.). 2-DG uptakewas measured using a fluorometric glucose uptake assay kit from Abcam(Cat. No. ab136956). As shown in FIG. 31, addition of eNampt increased2-DG uptake.

To determine the role of Nampt in Eno1 induced glucose uptake, humanskeletal muscle myotubes were treated with Eno1 for 48 hours asdescribed above, and the Nampt inhibitor FK866 was added 24 hours afterinitiation of Eno1 treatment, for a total FK866 treatment time of 24hours. 2-DG uptake was measured after 3 hours serum starvation using theAbcam glucose uptake assay kit described above. As shown in FIG. 32,Nampt inhibition by FK866 abolished Eno1 induced glucose uptake. Thisresult indicates that Nampt plays a role in Eno1 induced glucose uptake.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

INCORPORATION BY REFERENCE

Each reference, patent, patent application, and GenBank number referredto in the instant application is hereby incorporated by reference as ifeach reference were noted to be incorporated individually.

Description of Sequences SEQ ID NO: Sequence Description 1 DNA HumanEno1, transcript variant 1. 2 AA Human Eno1, transcript variant 1. 3 DNAHuman Eno1, transcript variant 2. 4 AA Human Eno1, transcript variant 2,also referred to as c-myc promoter-binding protein-1 (MBP-1). 5 DNAHuman Eno2. 6 AA Human Eno2. 7 DNA Human Eno3, transcript variant 1.Encodes isoform 1 of Eno3. 8 DNA Human Eno3, transcript variant 2.Encodes isoform 1 of Eno3. 9 AA Human Eno3, isoform 1. 10 DNA HumanEno3, transcript variant 3. Encodes isoform 2 of Eno3. 11 AA Human Eno3,isoform 2.

1. A pharmaceutical composition comprising a therapeutically effectiveamount of Eno1 or a fragment thereof.
 2. The pharmaceutical compositionof claim 1 for delivery to a muscle cell.
 3. The pharmaceuticalcomposition of claim 1, wherein the Eno1 comprises an Eno1 polypeptideor a fragment thereof.
 4. The pharmaceutical composition of claim 1,wherein the Eno1 comprises an Eno1 nucleic acid or a fragment thereof.5. The pharmaceutical composition of claim 1, wherein the compositionfurther comprises an expression vector encoding the Eno1 or fragmentthereof.
 6. The pharmaceutical composition of claim 1, wherein the Eno1or fragment thereof is biologically active.
 7. The pharmaceuticalcomposition of claim 1, wherein the Eno1 or fragment thereof has atleast 90% of the activity of a purified endogenous human Eno1polypeptide.
 8. The pharmaceutical composition of claim 1, wherein theEno1 is human Eno1.
 9. The pharmaceutical composition of claim 1,wherein the composition further comprises a microparticle.
 10. Thepharmaceutical composition of claim 1, wherein the composition furthercomprises a nanoparticle.
 11. The pharmaceutical composition of claim 1,wherein the composition further comprises an in situ formingcomposition.
 12. The pharmaceutical composition of claim 1, wherein thecomposition further comprises a liposome.
 13. The pharmaceuticalcomposition of claim 1, wherein the composition further comprises amuscle targeting moiety.
 14. The pharmaceutical composition of claim 13,wherein the muscle targeting moiety is a skeletal muscle targetingmoiety.
 15. The pharmaceutical composition of claim 13, wherein themuscle targeting moiety and the Eno1 polypeptide are in a complex. 16.The pharmaceutical composition of claim 15, wherein the Eno1 is releasedfrom the complex upon delivery to a muscle cell.
 17. The pharmaceuticalcomposition of claim 1, wherein the composition is formulated forparenteral administration.
 18. The pharmaceutical composition of claim1, wherein the composition is formulated for oral administration. 19.The pharmaceutical composition of claim 1, wherein the composition isformulated for intramuscular administration, intravenous administration,or subcutaneous administration.
 20. A method of decreasing blood glucosein a subject with elevated blood glucose, the method comprisingadministering to the subject a pharmaceutical composition comprisingEno1 or a fragment thereof, thereby decreasing blood glucose in thesubject.
 21. (canceled)
 22. A method of increasing glucose tolerance ina subject with decreased glucose tolerance, the method comprisingadministering to the subject a pharmaceutical composition comprisingEno1 or a fragment thereof, thereby increasing glucose tolerance in thesubject.
 23. (canceled)
 24. A method of improving insulin response in asubject with decreased insulin sensitivity and/or insulin resistance,the method comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof, thereby improvinginsulin response in the subject.
 25. (canceled)
 26. A method of treatingdiabetes in a subject, the method comprising administering to thesubject a pharmaceutical composition comprising Eno1 or a fragmentthereof, thereby treating diabetes in the subject.
 27. The method ofclaim 26, wherein the diabetes is type 2 diabetes or type 1 diabetes.28. The method of claim 26, wherein the diabetes is pre-diabetes. 29.(canceled)
 30. A method of decreasing an HbA1c level in a subject withan elevated Hb1Ac level, the method comprising administering to thesubject a pharmaceutical composition comprising Eno1 or a fragmentthereof, thereby decreasing the HbA1c level in the subject. 31.(canceled)
 32. A method of improving blood glucose level control in asubject with abnormal blood glucose level control, the method comprisingadministering to the subject a pharmaceutical composition comprisingEno1 or a fragment thereof, thereby improving blood glucose levelcontrol in the subject.
 33. (canceled)
 34. The method of claim 20,wherein glucose flux in a skeletal muscle cell of the subject isincreased.
 35. A method of increasing glucose flux in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof, thereby increasingglucose flux in the subject.
 36. (canceled)
 37. A method of increasingglycolytic activity or capacity in a skeletal muscle cell of a subject,the method comprising administering to the subject a pharmaceuticalcomposition comprising Eno1 or a fragment thereof, thereby increasingglycolytic activity or capacity in a skeletal muscle cell of thesubject.
 38. (canceled)
 39. A method of increasing mitochondrial freefatty acid oxidation in a skeletal muscle cell of a subject, the methodcomprising administering to the subject a pharmaceutical compositioncomprising Eno1 or a fragment thereof, thereby increasing mitochondrialfree fatty acid oxidation in a skeletal muscle cell of the subject. 40.(canceled)
 41. The method of claim 20, wherein the Eno1 is administeredparenterally.
 42. The method of claim 20, wherein the Eno1 isadministered orally.
 43. The method of claim 41, wherein the Eno1 isadministered by a route selected from the group consisting ofintramuscular, intravenous, and subcutaneous.
 44. The method of claim20, wherein the subject has any one or more of elevated blood glucose,decreased glucose tolerance, decreased insulin sensitivity and/orinsulin resistance, diabetes, elevated Hb1Ac level, and abnormal bloodglucose level control.
 45. The method of claim 20, further comprisingselecting a subject having any one or more of elevated blood glucose,decreased glucose tolerance, decreased insulin sensitivity and/orinsulin resistance, diabetes, elevated Hb1Ac level, and abnormal bloodglucose level control.
 46. The method of claim 20, wherein the subjectis human.
 47. A method for diagnosing an elevated blood glucose level ina subject, comprising: (a) detecting a level of Eno1 in a biologicalsample from the subject, and (b) comparing the level of Eno1 in thebiological sample with a predetermined threshold value, wherein a levelof Eno1 in the sample below the predetermined threshold value indicatesthe presence of elevated blood glucose in the subject.
 48. The method ofclaim 47, further comprising detecting the level of one or morediagnostic indicators of elevated blood glucose.
 49. The method of claim48, wherein the one or more additional diagnostic indicators of elevatedblood glucose is selected from the group consisting of HbA1c, fastingblood glucose, fed blood glucose, and glucose tolerance. 50-58.(canceled)
 59. The method of claim 47, wherein the presence of elevatedblood glucose in the subject is diagnostic of a disease or conditionselected from the group consisting of type 2 diabetes, pre-diabetes,gestational diabetes, and type 1 diabetes.
 60. The method of claim 47,further comprising administering a therapeutic regimen to the subjectwhen the presence of elevated blood glucose is determined, wherein thetherapeutic regimen is selected from the group consisting of drugtherapy and behavioral therapy, or a combination thereof. 61-64.(canceled)
 65. A method for monitoring elevated blood glucose in asubject, the method comprising: (1) determining a level of Eno1 in afirst biological sample obtained at a first time from a subject havingelevated blood glucose; (2) determining a level of Eno1 in a secondbiological sample obtained from the subject at a second time, whereinthe second time is later than the first time; and (3) comparing thelevel of Eno1 in the second sample with the level of Eno1 in the firstsample, wherein a change in the level of Eno1 is indicative of a changein elevated blood glucose status in the subject.
 66. The method of claim65, wherein the determining steps (1) and (2) further comprisedetermining a level of one or more additional indicators of bloodglucose selected from the group consisting of HbA1c level, fastingglucose level, fed glucose level, and glucose tolerance.
 67. The methodof claim 65, wherein the subject is treated with drugs for elevatedblood glucose prior to obtaining the second sample.
 68. The method ofclaim 65, wherein a decreased level of Eno1 in the second biologicalsample as compared to the first biological sample is indicative ofelevation of blood glucose in the subject.
 69. The method of claim 65,wherein an increased or equivalent level of Eno1 in the secondbiological sample as compared to the first biological sample isindicative of normalization of blood glucose in the subject. 70-72.(canceled)
 73. A kit for detecting Eno1 in a biological samplecomprising: (a) at least one reagent for measuring the level of Eno1 inthe biological sample; (b) a set of instructions for measuring the levelof Eno1 in the biological sample; and (c) a set of instructions fordetermining the level of blood glucose in the biological sample.
 74. Thekit of claim 73, further comprising at least one reagent for measuring alevel of HbA1c in the biological sample.
 75. The kit of claim 73,further comprising instructions for measuring at least one of HbA1clevel, fed blood glucose level, fasting blood glucose level, and glucosetolerance in the subject from which the biological sample was obtained.76-80. (canceled)